R Gay

V Della Marca

H Aziza

P Laine

A Regnier

S Niel

A Marzaki

Gate stress reliability of a novel trench-based Triple Gate Transistor

This paper addresses the reliability on a novel trench-based Triple Gate Transistor (TGT) fabricated in a 40nm embedded Non-Volatile Memory (e-NVM) technology. In the studied device, two vertical transistors are integrated in deep trenches alongside the main planar transistor to build a TGT. The reliability of this device is investigated targeting the gate oxide degradation, that represents one of the major reliability issues in e-NVM environment. Gate stress tests are motivated by the possibility to use independently trench transistors and the main planar transistor. Thus, the reliability of each gate oxide needs to be studied separately to the others. Moreover, different DC and AC stress tests are performed and analysed to understand the degradation mechanisms effecting the TGT device.

Introduction

Today, the semiconductor industry is dominated by submicron devices and unconventional device architectures designed for high density and performance enhancement [START_REF] Bohr | CMOS Scaling Trends and Beyond[END_REF]. In this context, several devices using multi-gate or gate-all-around transistors as an alternative to classical MOSFET have emerged in the last few years [START_REF] Ferain | Multigate transistors as the future of classical metal-oxide-semiconductor field-effect transistors[END_REF]- [START_REF] Colinge | Multiple-gate SOI MOSFETs[END_REF]. The studied device follows this trend: two lateral conduction channels controlled by vertical trenches filled with polysilicon are added to the main planar transistor. This arrangement results in a novel Triple Gate Transistor (TGT) which proposes an improved driving capability combined with a minimal footprint compared to conventional MOS transistors [START_REF] Gay | A Novel Trench-Based Triple Gate Transistor With Enhanced Driving Capability[END_REF]. However, the TGT fabrication process includes the etching of deep trenches and the growth of a thin oxide around the trenches profile. Thus, gate oxide stress reliability tests are carried out to assess the possibility of controlling individually trenches and planar transistors of the TGT through their respective gates. Usually, the oxide degradation investigation is based on DC stress tests. These experiments represent the worst case of the normal operating conditions. Concerning the device life aging in the e-NVM environment, AC stress tests have to be performed. In this paper both DC and AC stress methods are applied to the TGT for a deep comprehension. The first part of this work presents the TGT manufacturing process. Then, experimental DC and AC stress tests are presented and analysed.

Manufacturing and device description

The TGT is integrated in a Non-Volatile Memory environment and designed following the process flow of an embedded NOR Flash technology [START_REF] Niel | Embedded Select in Trench Memory (eSTM), best in class 40nm floating gate based cell: a process integration challenge[END_REF]. No additional steps or masks are required to fabricate the device. The TGT is manufactured as follows: active and oxide Shallow Trench Isolation (STI) regions are first performed (Fig. 1a). NVM implantation is then performed to realize the p-type well of the NMOS-based TGT. Then, the trench patterning presented in Fig. 1b is realized. An oxide is grown along sidewalls of the trenches which are filled with an in-situ doped polycrystalline silicon (Fig. 1c). To achieve the trench patterning, Chemical and Mechanical Polishing (CMP) is performed. As the TGT is realized in a NVM environment, its planar gate is processed based on the Flash floating gate stack (Fig. 1d). This latter includes two shorted polysilicon layers, an oxide/nitride/oxide inter-poly dielectric and a tunnel oxide. The planar gate contact is realized through the gate stack. Two vertical trenches contacts are shifted with respect to the gate stack and connected to the poly-silicon gate trenches. Finally, source and drain contacts are formed with n+ implantations.

Fig. 2a shows the TGT top view. The first conduction channel located between the Drain and the Source is controlled by the planar stacked gate (MG). This transistor is represented with a solid line. The second and third channels, also located between the Drain and the Source, are controlled by the gate trenches (shorted). These devices denoted by MOS Trenches (MT) transistors are represented in dotted line. Finally, the TGT has three conduction channels and it can be represented by three MOS transistors connected in parallel.

Fig. 2b and Fig. 2c show TEM cross-section views of the TGT in width (W) and length (L) directions respectively. Based on these figures, we can see that the proposed 3D structure can be used to build an area-efficient transistor with a high driving capability compared to classical planar MOS transistor [START_REF] Gay | A Novel Trench-Based Triple Gate Transistor With Enhanced Driving Capability[END_REF].

Experimental results

Experimental setup

The TGT is made of two different gates: MG controls the planar transistor and MT controls the vertical trenches. Thus, stress tests will address the reliability of both gate oxides. The tested samples, manufactured in a 40nm e-NVM technology, are integrated in a 300 mm wafer. The experimental setup includes an Agilent 4156C semiconductor parameter analyser for electrical characterizations coupled with a Keysight 33500B waveform generator using a Keithley 7174A switch matrix. All stress tests and measurements are performed at room temperature (25°C).

AC stress tests are defined with the following parameters: a rising/falling time (trf) of 100 ns, a period (cycle) of 200 µs with a 50% duty cycle. DC and AC stress tests are performed under positive gate bias with drain, source and bulk nodes connected to ground. Tests are periodically interrupted and drain current versus gate voltage (Id-Vg) characteristics are extracted for both MG and MT transistors to evaluate device performances. A gate voltage sweep ranging from 0 V to 3 V with a drain voltage (Vd) at 0.7 V is used. The threshold voltage (Vt) extraction is performed for a gate voltage associated to a drain current equal to 100 nA. The Subthreshold Slope (Ss) is defined as the required voltage to increase the current by one decade between 1 nA and 10 nA.

DC stresses

Preliminary measurements are carried out to evaluate the optimal gate bias stress for each transistor. Fig. 3. presents the gate current evolution after applying a Linear Ramp Voltage Stress (LRVS) to the MT and MG gates. We can see that the MG oxide breakdown is reached at 15 V and the MT one at 10 V. Note that MT gate current starts increasing before the MG one. This early leakage current is caused by an irregular thickness of the trench oxide which can be explained by the nonuniform oxide growth along the sidewall. This means that trenches and planar gate cannot support the same bias stress, resulting in different AC and DC test conditions: MG gate stress is performed at VG = 10 V and the MT one at VG = 8 V. Constant Voltage Stress (CVS) performed on MG (Fig. 4a) and MT (Fig. 4b) transistors are shown. Note that in Fig. 4a, during the VgMG bias stress, VgMT = 0 V and during the VgMT bias stress, VgMG = 0 V. For the MG gate stress, we can see a progressive positive shift of the Vt versus stress time and a degradation of the Ss. However, no significant variation is observed for the MT transistor up to 100s (Fig. 4b). At 300s, a flat Id-VgMT characteristic is measured showing that the transistor breakdown has been reached.

AC stress

Fig. 5a shows the Id-VgMG characteristics during AC stress tests for VgMG = 10 V. As the number of cycles increases up to 3•10 6 , Vt and Ss degradation and the leakage current (Ioff) extracted at VgMG = 0 V increases. Fig. 5b presents Id-VgMT characteristics for VgMT = 8V AC stress. We can notice a slight decrease of driving current (Ion) extracted at 3V gate voltage up to 570k cycles. The MT results off after 1.3M cycles.

MG transistor versus MT transistor reliability

In some low voltage or analog applications, one can use the different transistors of the TGT separately. The objective here is to study the impact of the MT gate stress on the MG performances and conversely. During the transistor stress test, the other transistor is grounded. This reliability evaluation is conducted by extracting and analysing I-V characteristics as well as key performance parameters (Ioff, Ion, Ss and Vt) after CVS and AC stress.

Impact of MG stress on MT performances

The Vt and Ss shifts (∆Vt and ∆Ss) versus stress time are presented after CVS (Fig. 6a) and AC stress (Fig. 6b) applied to the MG gate. For a fair comparison between AC and CVS tests, the effective stress time is plotted (i.e., the relaxation time of AC stress is not accounted). The MG transistor ∆Vt (∆VtMG) increases with stress time from 10 s for both AC and CVS tests while the MT transistor ∆Vt (∆VtMT) remains constant. The variation of ∆VtMG is explained by the electron trapping at the channel/oxide interface and in the oxide bulk [START_REF] Tan | Application of the difference subthreshold swing analysis to study generation of interface traps in MOS structures due to Fowler-Nordheim aging[END_REF]. The ∆Ss shows the same trend. It increases only for the MG transistor since 30 s stress which indicates interface states generation [START_REF] Dimaria | Impact ionization, trap creation, degradation, and breakdown in silicon dioxide films on silicon[END_REF] more present in AC compared to CVS stress. The ∆VtMT and ∆SsMT remain constant during the stress time suggesting that the MT transistor is not impacted by charge trapping occurring during MG gate stress.

The Ion and Ioff variations (∆Ion and ∆Ioff) after CVS (Fig. 7a) and AC (Fig. 7b) stress applied to the MG gate are reported. For both stresses, ∆Ion decreases during the time for the MG (∆IonMG) while it remains constant for the MT (∆IonMT). No significant ∆Ioff is measured for both transistors and stress types. These variations can be explained by trapped charge or interface traps generation, localized at channel-oxide tunnel interface [START_REF] Dimaria | Impact ionization, trap creation, degradation, and breakdown in silicon dioxide films on silicon[END_REF]. During the I-V reading state, interface traps become negatively charged and create an inversion region. This region leads to increases the drain resistance, resulting in a decreases of the driving current Ion and increases of the Ss parameter.

Impact of MT stress on MG performances

We report here the evolution of ∆Vt and ∆Ss after CVS (Fig. 8a) and AC (Fig. 8b) stress tests applied to the MT gate. In the case of the CVS, ∆Vt and ∆Ss of both transistors remain constant until 200 s. After 200 s, ∆VtMT and ∆SsMT significantly increase, which corresponds to the MT transistor breakdown. In contrast, ∆VtMG and ∆SsMG decrease, indicating that the MG transistor is damaged but still operating. However, for the AC stress presented in Fig. 8b, a different behaviour is observed. ∆VtMT increases and ∆SsMT decreases at 100 s while ∆VtMG and ∆SsMG are not affected. Therefore, the MG transistor is not damaged during MT transistor AC stress. Fig. 9a presents ∆Ion and ∆Ioff evolution versus stress time during the MT gate CVS and the Fig. 9b shows the variation for the AC stress. In the first case, ∆IoffMT increases and ∆IonMT decreases since 200 s. The MG transistor is also impacted with a slight variation of its parameters (∆IonMG and ∆IoffMG). In Fig. 9b the ∆IonMT starts decreasing just before 50 s while no shifts are observed for the MG transistor parameters. These results indicate that the MG transistor is not damaged during MT gate AC stress.

Summary results

Fig. 10a reports the MT transistors Id-VgMT characteristics after the MG gate stress for different stress methods. The Id-VgMT characteristics are similar before and after the different stress types. Thus, the MG degradation does not damage the MT transistor, whatever the stress is. Regarding the fact that for the LRVS stress, the MG transistor breakdown does not impact the MT transistor. It can be related to the MG oxide breakdown probability which depends on its area [START_REF] Wu | Weibull slopes, critical defect density, and the validity of stress-induced-leakage current (SILC) measurements[END_REF]. Indeed, the intersection between the MG oxide and the MT oxide located at the substrate-trench-gate intersection region is narrow, limiting the impact of the MG stress on the MT.

In Fig. 10b where different stress types are applied to the MT gate, the MG transistor is damaged. Specifically, after the LRVS test, the MG transistor is no operational (the drain current is constant at 10 µA) suggesting that the MG transistor has been damaged by the MT breakdown. After the CVS test, the final characteristic shows a low Ion and a high Ioff current compared to the fresh characteristic. Finally, the AC stress test has no impact compared to the fresh characteristic except a lower Ion current. The difference between the characteristics after AC stress and LRVS can be explained by the charge detrapping during the relaxation time [START_REF] Ershov | Transient effects and characterization methodology of negative bias temperature instability in pMOS transistors[END_REF] (i.e, stress interruption during the Id-VgMG extraction). However, after the AC stress the I-V characteristic is comparable to the fresh one as it includes periodically stress interruptions defined by the AC duty cycle.

The impact of the MT stress on the MG transistor degradation needs more investigation. What we can infer from Fig. 10b is that the LRVS test of the MT transistor short-circuits the source and drain of the MG transistor, resulting in a high current (10 µA) when its gate voltage is equal to zero. Moreover, due to the manufacturing process, we have observed a tip effect located at the substrate/trench/gate intersection oxide region which can lead to an enhanced electrostatic potential. Therefore, this effect can explain the non-reciprocal oxide breakdown between the MG and MT transistor.

Conclusion

In this paper we investigate the electrical reliability of a novel TGT transistor under positive gate DC and AC stresses. The TGT is made of distinct transistors (MG and MT) controlled by independent gates. The electrical parameters evolution as a function of stress time revealed that the MG gate degradation does not impact the MT transistor, whatever the stress type applied. On the contrary, the MT gate degradation leads to the MG transistor damage depending on the applied stress type. Indeed, DC stress applied to the MT gate strongly damage the MG, but AC stress has a minor impact as a slight decrease of the Ion parameter of the MG transistor is reported. Therefore, for decorrelated gate applications, it will be suitable to report the maximal stress on the MG transistor.

Fig. 1 .Fig. 2 .

 12 Fig. 1. TGT process flow. a) Active/STI definition, b) trenches etching, c) oxide grown and polycrystalline silicon deposition, d) gate stack patterning.

Fig. 3 .

 3 Fig. 3. I-V characteristic of LRVS applied to MG gate and MT gate (three samples).

Fig. 4 .

 4 Fig. 4. DC gate bias stress applied on a) MG for VgMG=10V and b) on MT for VgMT=8V.

Fig. 5 .

 5 Fig. 5. Id-Vg characteristics in AC stress mode a) MG stressed with VgMG = 10 V, Vd,s,b = 0 V and (b) MT stressed with VgMT = 8V, Vd,s,b = 0 V.

Fig. 6 .

 6 Fig. 6. Vt and Ss shifts as function of (a) CVS and (b) AC stress time applied to the MG gate (VgMG = 10 V, Vd,s,b = 0 V).

Fig. 7 .

 7 Fig. 7. Ion and Ioff shifts as function of a) CVS and b) AC stress time applied to the MG gate (VgMG = 10 V, Vd,s,b = 0V)

Fig. 8 .

 8 Fig. 8. a) CVS and b) AC stress dependence of Vt and Ss shift as function of stress time for MT gate stress with VgMT= 8 V, Vd,s,b = 0 V.

Fig. 10

 10 Fig. 10. a) Id-VgMT curves versus MG stress and b) Id-VgMG curves versus MT stress obtained after LRVS, CVS and AC stress.


Samuel Alizon
email: samuel.alizon@cnrs.fr

Paul E Turner
email: paul.turner@yale.edu

Can we eradicate viral pathogens?

The COVID-19 pandemic has led to a resurgence of the debate on whether host-parasite interactions should evolve towards avirulence. In this review, we first show that SARS-CoV-2 virulence is evolving, before explaining why some expect the mortality caused by the epidemic to converge towards that of human seasonal alphacoronaviruses. Leaning on existing theory, we then include viral evolution into the picture and discuss hypotheses explaining why the virulence has increased since the beginning of the pandemic. Finally, we mention some potential scenarios for the future.

The hope of treatments and (RNA) vaccines

The popularisation of vaccination since the end of the XIXth century and the discovery of therapeutic drugs against bacterial, fungal, and protozoan infections in the early XXth century led to the hope that the burden caused by infectious diseases could be ended. This culminated in the 1960s with the infamous, but most likely apocryphal, quote from a surgeon general of the United States stating that it was "time to close the book on infectious diseases" [START_REF] Spellberg | On the exoneration of Dr. William H. Stewart: debunking an urban legend[END_REF]. The emergence or re-emergence of viral threats, starting with HIV but more recently with Ebola, Chikungunya, and Zika viruses in humans and foot-and-mouthdisease virus and begomoviruses in agricultural animals and plants, as well as the generalisation of drug resistance mutations, shattered this optimistic view. Furthermore, because of economic constraints, parasites always remained a major threat in many countries of the world.

Somehow paradoxically, the current COVID-19 pandemic is giving rise again to the hope of eradicating infectious diseases. This is largely motivated by the success in discovering, manufacturing, and implementing safe and efficient SARS-CoV-2 vaccines in less than a year. That some of the most widely-used of these vaccines rely on newer RNA technology further stimulates optimistic predictions. From a history of science perspective, the view traces back to Paul Ehrlich's "magic bullet" reference to highly-accurate therapies, since RNA vaccines can be designed to target specific motifs. Their production is also much more rapid than traditional protein-based vaccines. Their implementation is perhaps the only negative point, with a current necessity to store RNA vaccine doses at -80°C, which is prohibitively expensive in many healthcare settings.

Are RNA vaccines fundamentally changing the equation? Is the bold 1960's-era vision of eradicating diseases now within our grasp? Evolutionary biologists and ecologists can bring valuable insights based on decades of experience analysing how microbes adapt to anthropic 1 perturbations. In fact, the main reason why RNA vaccines were needed so rapidly is probably due to a virus spillover from an animal reservoir into the human population, a phenomenon that has been studied by evolutionary ecologists [START_REF] Lloyd-Smith | Epidemic Dynamics at the Human-Animal Interface[END_REF].

As illustrated by this special issue Virus evolution on the mutualist -parasite continuum, it is important to bear in mind that virus population have a huge diversity, a wide array of potential hosts, and that they are not always pathogenic. Furthermore, as also stressed by contributions in this issue, viruses evolve rapidly, implying that they can readily colonise new hosts or face changing environmental conditions. Importantly, by doing so, they modify the selective pressures that act on their evolution. In other words, public health policies should not be designed to target current viruses but rather evolving viruses and this is why our community has a role to play.

2 Defining 'pathogens' can be difficult First, it is important to stress that the notion of pathogen is itself somehow loose [START_REF] Méthot | What is a pathogen? Toward a process view of host-parasite interactions[END_REF]. This is illustrated by the title of the special issue, itself inspired by an article from Paul W. Ewald [START_REF] Ewald | Transmission modes and evolution of the parasitism-mutualism continuum[END_REF]. Obviously, all parasites can harm their hosts, otherwise they would not be called as such. But the phenotype is the result of the parasite genotype, the host genotype, and the environment [START_REF] Shapiro | Evolution of mutualism from parasitism in experimental virus populations[END_REF]. For instance, some HCV mutations appear to only be detrimental when infecting human hosts with a specific mutation [START_REF] Ansari | Genome-to-genome analysis highlights the effect of the human innate and adaptive immune systems on the hepatitis C virus[END_REF]. It is also evident that context matters. A classic example in humans is that coinfection by a flavivirus (GB virus C) or by herpesviruses (HHV-6 or HHV-7) can reduce HIV infection virulence [START_REF] Virgin | Redefining chronic viral infection[END_REF]. In plants, infection by one virus species can protect against infection by those from another virus family, a phenomenon known as cross-protection [START_REF] Ratcliff | Gene Silencing without DNA: RNA-Mediated Cross-Protection between Viruses[END_REF]) that can also be interpreted as a priorinfection pattern in epidemiology [START_REF] Sofonea | Exposing the diversity of multiple infection patterns[END_REF]. Even at the cellular level, viruses have been shown to compete for resources making virulence (or pathogenicity) difficult to define in a biologically diverse context [START_REF] Turner | Escape from prisoner's dilemma in RNA phage $\Phi 6$[END_REF][START_REF] Alizon | Multiple infections and the evolution of virulence[END_REF]. From a more mundane perspective, it is unclear whether a current day 'pathogen' might be useful somehow in the future, e.g., as a means to implement biological control of another parasite, or to yield genes beneficial for an applied use. Furthermore, we keep discovering new viruses. SARS-CoV-2 is of course an extremely deadly example, but many of these new viruses are only known through their DNA found in metagenomes. As illustrated by the study of Zhao et al. in this special issue, analysis of eukaryotic genome sequence data can be used to detect circular replication-associated protein-encoding single-stranded (CRESS) DNA viruses, thereby expanding discovery of previously-unknown virus diversity.

For simplicity, in the following sections we will focus on viruses that are more obviously deleterious to the host, so that virus pathogenicity is less questioned. Even here, the eradication question can be considered in a practical way (is eradication feasible?) and in an ethical way (should this eradication be attempted?).

Eradication requires will (and funding)

Regarding the practicality, biotechnology increasingly allows the generation of viruses in vitro based on their genomic sequence information alone.

Setting aside for the moment the ethics of such engineering, we note that treatment availability is not enough to guarantee eradication at a population level (at the individual level, several treatments or vaccines do achieve this goal). As illustrated with SARS-CoV-2, although vaccines decrease the probability to be reinfected, their main efficacy is against severe disease [START_REF] Creech | SARS-CoV-2 Vaccines[END_REF]. Even with an infection-blocking vaccine, implementation and surveillance require original approaches, as illustrated in the (successful) case of variola virus (smallpox disease) elimination [START_REF] Henderson | The eradication of smallpox -An overview of the past, present, and future[END_REF]. Conversely, for HPV, safe and efficient vaccines have been implemented for over a decade and the most oncogenic types (HPV16 and HPV18) are nowhere near extinction at a worldwide scale [START_REF] De Martel | Worldwide burden of cancer attributable to HPV by site, country and HPV type[END_REF]. Recent results from the UK reporting that HPV vaccination decreased cervical cancer risk by 72 to 94% in women above 30-years old that were vaccinated at age 12 or 13 [START_REF] Falcaro | intraepithelial neoplasia incidence: a register-based observational study[END_REF] confirm that vaccine efficacy is not to blame; rather, the lack of funding and of political will seem to be the main factors.

The contrasting cases of HPV and variola virus demonstrate the biological and economical limitations of vaccination. These shortcomings are also visible in the terminology and the distinction between control, disease elimination, infection elimination, and eradication. In the case of the ongoing pandemic, some countries have so far succeeded in greatly reducing SARS-CoV-2 infections; the development of treatments leads to the hope of eliminating the disease (COVID-19), but most countries mainly focus on controlling the infection. Eradication currently seems unrealistic given the vaccine efficiency in blocking infections and the possibility for presence in animal reservoirs, which could differentially impact virus evolution. For instance, differences in host-cell receptors can drive divergence among influenza virus lineages as they adapt in vitro Barnard et al.. The host itself is only part of the equation and for an ssRNA plant virus the habitat in which these plants are located also shapes evolutionary dynamics Peláez et al..

Viruses evolve

Under-appreciating virus evolution jeopardizes the promise and success of eradication programs. Similarly, before evolution of generalised drug resistance, the World Health Organisation was aiming for eradication of malaria Plasmodium parasites [START_REF] Molyneux | Disease eradication, elimination and control: the need for accurate and consistent usage[END_REF]). In a virus example, the main concern in poliovirus eradication is that epidemics are now caused by evolutionary reversion of the oral vaccine strains, illustrating unwarranted (long-term) effects of eradication policies [START_REF] Famulare | Assessing the stability of polio eradication after the withdrawal of oral polio vaccine[END_REF].

Theory in evolutionary ecology tells us a lot about population extinctions. For instance, the closer a population approaches extinction, the stronger the selective pressure for 'evolutionary rescue' [START_REF] Gonzalez | Evolutionary rescue: an emerging focus at the intersection between ecology and evolution[END_REF]. If we transpose this to viruses, the more likely an eradication policy is to succeed, the more we should beware of an evolutionary (phenotypic) response in the pathogen. This evolution response can manifest itself in several ways. The most common one is virus resistance to the intervention, which means that we lose a treatment. Unfortunately, as demonstrated in the case of Marek's disease virus (MDV) in poultry farming [START_REF] Read | Imperfect Vaccination Can Enhance the Transmission of Highly Virulent Pathogens[END_REF], vaccination may exert selection that favours more virulent viral strains. Fortunately, such evolutionary response is less frequent for prophylactic vaccines, i.e., administered before infection, than for curative treatments given to infected hosts because in the former case the population within-host diversity remains limited [START_REF] Kennedy | Why does drug resistance readily evolve but vaccine resistance does not?[END_REF]. A potential worry, however, is that treatments targeting more-virulent strains can lead to the correlated evolution of both increased resistance and virulence (Alizon, 2020).

Interestingly, the evolution of pathogen resistance issues that are faced by clinicians in hospital are also evidenced in parasites of bacterial populations. For instance, the study by [START_REF] Common | Diversity in CRISPRbased immunity protects susceptible genotypes by restricting phage spread and evolution[END_REF] in this special issue shows how bacteria have evolved a diversity of defence mechanisms, which select for bacteriophage ability to evolve counter-resistance.

Not a call for inaction

The threat of virus evolution should in no way serve as an excuse for inaction. First, the potential risks need to be assessed. Second, it is important to mitigate these risks as much as possible. Again, the SARS-CoV-2 pandemic raised these questions in a particularly acute way. The development of RNA vaccines was undoubtedly fast and, when they were first implemented at the end of 2020, clinical studies had somehow limited overview on potential complications (which happened to be as rare as suggested by the initial results). However, the very high fatality rate of the infection (at least 10 times that of seasonal influenza) combined with its massive spread clearly outweighed the uncertainty risk. Furthermore, as pointed out by Alizon and Sofonea in their review in this issue, SARS-CoV-2 virulence has increased since the beginning of the epidemic in the absence of vaccination, showing that inaction does not guarantee virus evolution will improve the situation.

One possibility of action is to devise 'evolution-proof' treatments. It can be debated whether these can ever truly exist because, as in the case of anti-microbial peptides which were initially thought to be extremely robust to the evolution of resistance [START_REF] Perron | Experimental evolution of resistance to an antimicrobial peptide[END_REF], microbes may always find their way. However, the fact remains that anticipating an evolutionary response should help to slow it down. For instance, at the Jacques Monod conference associated with this special issue, we learned how genetic engineering can radically decrease the aforementioned risk of reversion in the polio vaccine [START_REF] Yeh | Engineering the Live-Attenuated Polio Vaccine to Prevent Reversion to Virulence[END_REF].

Evolutionary biologists have much to contribute to the elaboration of eradication, elimination, or control strategies. However, when it comes to human health, we should also bear in mind that this is not as simple as writing an opinion piece in a society journal. In our special issue, Froissart and Brives use the example of phage therapy to stress how our community needs to navigate and interact with regulation of medicines, so that promising new therapies actually have an impact.

The ongoing pandemic has led to the realisation that mutations can generate 'variants' directly responsible for national lock-downs and reduced global travel. This is perhaps one of the strongest illustrations of the importance of virus evolution in recent history. We also know that public health policies will act as a selective pressure on viruses. This should by no means be a call for inaction. However, it is vital to monitor phenotypic evolution (Kennedy and Read, 2020) and to be at least one step ahead of viruses when implementing any type of control strategy.

Acknowledgements

This special issue stems from the Jacques Monod conference Virus evolution on the mutualistparasite continuum that took place in Roscoff (France), on October 21-25, 2019. Little could we know that SARS-CoV-2 was probably emerging in the human population at the time.


Cosette Rebouissou

Marie-Odile Baudement

Thierry Forné
email: forne@igmm.cnrs.fr

The High-salt Recovered Sequence-sequencing (HRS-seq) method: exploring genome association with nuclear bodies

Keywords: High-order chromatin organization, nuclear bodies, Quantitative PCR HRS-seq

Recent works indicate that, at specific loci, interactions of chromatin with membraneless organelles self-assembled through mechanisms of phase separation, like nuclear bodies, are crucial to regulate genome functions, and in particular transcription. Here we describe the protocol of the High-salt Recovered Sequence sequencing method whose principle relies on high-throughput sequencing of genomic DNA trapped into large RNP complexes that are made insoluble by high-salt treatments.

Introduction

The importance of genome organization at the supranucleosomal scale in the control of gene expression is increasingly recognized today [START_REF] Court | Modulated contact frequencies at gene-rich loci support a statistical helix model for mammalian chromatin organization[END_REF][START_REF] Ea | Contribution of Topological Domains and Loop Formation to 3D Chromatin Organization[END_REF]. In mammals, Topologically Associating Domains (TADs) and the active/inactive chromosomal compartments are two of the main nuclear structures that contribute to this organization level. However, recent works indicate that, at specific loci, chromatin interactions with nuclear bodies could also be crucial to regulate genome functions, in particular transcription (3). They moreover suggest that these nuclear bodies are membrane-less organelles dynamically selfassembled and disassembled through mechanisms of phase separation. Based on previous experimental approaches developed in our laboratory (4,5), we have recently developed a novel genome-wide experimental method, High-salt Recovered Sequences sequencing (HRS-seq), which allows the identification of chromatin regions associated with large ribonucleoprotein (RNP) complexes and nuclear bodies (6). . Transcriptionally active nuclei preparations are treated at a high salt concentration and the insoluble fraction is purified and sequenced (Figure 1). We argue that the physical nature of such RNP complexes and nuclear bodies appears to be central in their ability to promote efficient interactions between distant genomic regions. The development of novel experimental approaches, including our HRS-seq method described in the protocol below, is opening new avenues to understand how self-assembly of phase separated nuclear bodies contributes to mammalian genome organization and gene expression.

Methods

Cell nuclei preparation (see Note 6)

1.

-If working with non-adherent cultured cells, proceed with step 2 below.

-If working with adherent cell cultures, trypsinize, wash and filter through 40m cell strainer to make a single-cell suspension before proceeding with step 2 below.

2.

Wash cells in PBS.

3.

Resuspend cells in 1.5 ml buffer 1.

4. Add 0.5 ml buffer 2 and mix slightly.

5.

Put on ice 2 or 3 minutes.

6. Put 1 ml in 14 ml tubes (x2 tubes) containing 4 ml of buffer 3.

7.

Centrifuge 20 minutes at 11300 g, 4°C.

8.

Remove supernatant (use several tips to avoid NP40 contamination of the nucleus pellet).

9.

Resuspend pellets in 50 µl of glycerol buffer and take a 4 µl aliquot to count nuclei in a Thoma cell. Adjust to a final concentration of about 20 million nuclei in 100 µl and, from this stock, make 100µl aliquots containing 150,000 nuclei diluted in glycerol buffer. Freeze into liquid nitrogen and store at -80°C.

Generation of nuclear halos by high-salt treatment

10. Thaw on ice an aliquot containing 150,000 nuclei in a 1.5ml Eppendorf tube (important: do not increase the number of nuclei treated during these steps i.e.

150,000 nuclei, see Note 7).

11. For 100µl of aliquot, add 900µl of Triton buffer on the nuclei and homogenize by pipetting.

12. Incubate 15 minutes on ice (mix gently by pipetting 3 times during incubation).

13. Incubate 20 minutes at 37°C in a water bath or equivalent apparatus.

14. Transfer the Eppendorf content onto the filter of a centrifugal filtration unit equipped with a 12-ml collector tube below.

15. Push 2 drops out with the help of the piston or Home-Made Apparatus (HMA) (see Note 2).

16. Apply again a pressure with the help of the piston or HMA to push the liquid through the filter at a drop-by-drop rhythm (no more than 1 drop per second)

until as little liquid remains as possible above the filter (do not dry the filter).

17. Add 1ml of extraction buffer, homogenize gently by pipetting and push 6 drops out (dead volume of the column).

18. Incubate 5 minutes at room temperature.

19. Use the piston (or HMA) to pass the entire volume of extraction buffer through the filter at a drop-by-drop rhythm.

20. Add 950 µl of enzyme buffer. Pass all the liquid through the filter at a drop-bydrop rhythm. Repeat three more times, passing all the liquid through the filter and emptying the collection tube to the waste (these washings allow removal of high salt concentration presents in the extraction buffer).

Digestion of nuclear halos

21. For each filtration unit, add a mix containing 960µl of enzyme buffer and 20µl 10U/µl StyI, and homogenize gently by pipetting (see Note 8).

22. Add a band of parafilm under the filter part to obstruct the filtration unit and place it back on the collector tube.

23. Incubate the filtration unit for 30 minutes at 37°C (take care that all the liquid in the filtration unit should be immersed into the water bath and check regularly that there is no leakage due to a lack of parafilm obstruction). 27. Add 1ml of enzyme buffer to rinse the nuclear halos and pass it all through the filter in the same collector tube as step 26. Save this eluate, corresponding to the soluble fraction (2ml).

28. Add 500µl of enzyme buffer to the filter, place a parafilm to obstruct the filtration unit and put the filtration unit on a clean collector tube (12 ml).

29. Add 500µl of 2X PK buffer and 10µl of 20 mg/ml proteinase K to the filter.

Homogenize gently by pipetting.

30. Add 200µL of 10X PK buffer and 10µl of 20 mg/ml proteinase K to the soluble fraction from step 27. Homogenize gently by pipetting.

31. Incubate the filtration unit and the tube containing the soluble fraction for 30 minutes at 50°C in a water bath (take care to keep them well immersed).

32. Pass all the liquid through the filter of the filtration unit. Save this eluate in a clean collection tube, corresponding to the insoluble fraction (1ml).

Steps 33-38 refer to the Macherey-Nagel® NucleoSpin Clean-up® protocol.

33. Add 5ml of NTB buffer to the insoluble fraction and 11ml to the soluble fraction

(5 volumes). The soluble and insoluble fractions are then processed in parallel.

34. Add 700µl of one fraction to a NucleoSpin column and pass all the liquid by aspiration on a vacuum manifold (or by centrifugation 30 seconds at 11,000g, room temperature). Repeat these 700µl loadings until all of one fraction has passed through the same column.

35. Pass 700µl of NT3 buffer through the column using vacuum manifold (or centrifuge 30 seconds at 11,000g, room temperature) and empty collector tube in the waste.

36. Pass again 700µl of NT3 buffer through the column as in step 35.

37. Place the column on a clean Eppendorf tube properly labelled.

38. Add 50µl of NE buffer. Incubate 1 minute minimum at room temperature.

Centrifuge 1 minute at 11,000g, room temperature.

For each sample, repeat steps 10-38 until a total of at least 150 ng of DNA is obtained for each fraction (DNA quantification by Qubit or an equivalent apparatus).

Add milli-Q water to each fraction to a final volume of 500µl (see note 9).

Re-digestion of genomic DNA (see

Notes

1. DTT, PMSF and Aprotinin should be added extemporaneously to this buffer.

2. Home Made Apparatus (HMA) for pressurizing filtration units. A home-made apparatus was developed for pressurizing filtration units. Such apparatus is making use of a hydrostatic pump. It allows to exert a fixed and constant pressure on the liquid above the filter which can be easily set-up by controlling the speed of the hydrostatic pump (see Figure 2).

3. We recommend to use homemade restriction buffer since commercial buffers usually contain reducing agents like DTT that may alter the insoluble fraction thus affecting DNA retention.

4. The two primers used to make the adaptors are mixed in equimolar amount and annealed together on a PCR bloc by heating them at 100°C and then decreasing the temperature to 25°C by 0.1°C steps thanks to 750 cycles of 1 minute each.

5. Some enzyme suppliers provide MmeI directly supplemented with SAM (e.g.

NEB)

. In that case SAM is replaced by 10 µl of milli-Q water (Step 52).

6. The present protocol is intended for cultured cells as previously described (9).

If working with tissues, prepare nuclei as previously described in references (7,8) 12. Critical step: 150ng corresponds to the amount recommended to ensure a good quality of the final libraries. Add the biotinylated adaptor and the other reagents to the genomic DNA as specified. Do not make a "pre-mix" of the regents with the ligase as this may lead to the formation of dimers of adaptors and would reduce the quality of the sequencing library.

13. This section provide guidelines to data processing as used in our previous work (6). The choice of software used to perform these tasks are largely depended on laboratory facilities and experimental conditions.

  26. PCR master mix (48 µl per reaction, final concentrations given for 1X mixture, after addition of 2µl template in step 58): 0.5µl of 2U/µl Phusion High-Fidelity DNA polymerase (1U final), 36µl of milli-Q water, 10µl of 5X HF Phusion buffer, 0.5µl of 25µM GEX PCR primer 1 (250nM final) (5'-CAAGCAGAAGACGGCATACGA-3'), 0.5µl of 25µM GEX PCR primer 2 (250nM final) (5'-AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA-3'), 250µM dNTPs. 27. PCR cycler. 28. Electrophoresis equipment and UV reader or DarkRider. 29. 1X NEBuffer 2 (10X stock from NEB): 10mM Tris-HCl pH 7.9, 50mM NaCl, 10mM MgCl2. 30. Costar Spin-X centrifuge tube filters (45µm). 31. 20 mg/ml Glycogen. 32. 3M Sodium acetate pH 5.2. 33. Absolute ethanol and 70% ethanol.34. 2100 BioAnalyzer (Agilent) with high sensitivity DNA Chips. 35. Next-generation sequencing machine (e.g. Illumina Hi-Seq 2000).

24 .

 24 Collect evaporation drops in the tube cap and around tube walls and place them back in the liquid onto the filter. Add 20µl of 10U/µl StyI, mix gently by pipetting and incubate 30 minutes more.3.4 DNA purification ofStyI-digested DNA from soluble and insoluble fractions 25. Remove tubes from the water bath. Collect evaporation drops in the tube cap and around the tube walls, and place them back with the remaining liquid on the filter. Homogenize gently by pipetting. 26. Remove the parafilm, collect residual drops on the parafilm and place them back in the liquid on the filter. Place the filter part on a clean collector tube and pass all the liquid through the filter.

14 . 15 .

 1415 According to our protocol, Mme I digestion provides reads of 18 to 20 nucleotides. According to our protocol, the reads should have one of their extremity next to a StyI restriction site.

Figure caption:Figure 1 : 18 Figure 2 :

 1182 Figure caption:

  FIGURE 1

  and proceed with step 2.

	if less than 500ng have been obtained after step 40 (final volume of 50µl i.e.
	10ng/µl), the sample will need to be concentrated to at least 10ng/µl by
	ethanol precipitation (to a 50 µl sample add 2,5 µl of 5M NaCl, 1µl of 20mg/ml
	glycogen and 150µl of ethanol, mix, keep 45 minutes at -80°C and centrifuge
	15 minutes at 15,700g, room temperature). If more than 500ng have been
	obtained, the sample can be used directly without the concentration step 41.
	7. Important note: do not increase the number of nuclei treated during steps 10-
	38. It is extremely important to keep a small number of nuclei (150,000) diluted
	in a big volume during these steps because nuclear halos tend to aggregate
	together and such aggregation could generate in vitro artefacts. For the same
	reason, do not pellet nuclear halos by centrifugation, but, as stated in this
	protocol, always use filtration units and apply gentle pressures on the liquids
	using a piston or HMA (10).
	8. If using a different enzyme to StyI, the enzyme buffer and the Biotinylated
	adaptor 1 provided in this protocol will need to be modified accordingly.
	9. Quality controls can be made at this stage by qPCR to check for enrichment of
	specific sequences (4, 6).
	10. Partial digestion is a technical concern; steps 39-41 ensure full digestion
	before sequencing library preparation.
	11. The minimal concentration for DNA samples after step 40 is 10ng/µl. At this
	concentration, one requires to add 15µl (150ng) at step 42, which is the
	maximum volume allowed to reach 50µl of final volume at step 43. Therefore,

Acknowledgements

We thank Mathilde Tancelin for help in improving the HRS assay, Jean Casanova for developing the HMA system, Laurent Journot and all the engineers of the MGX platform (Montpellier) who helped to design HRS-seq HRS-seq 16 DNA libraries. This work was supported by grants from the Agence Nationale de la Recherche (CHRODYT, ANR-16-CE15-0018-04), the AFM-Téléthon (N° 21024) and the Centre National de la Recherche Scientifique (CNRS).


Julie Guillemant

Alexandra Berlioz-Barbier

Jérémie Ponthus

Luis P De Oliveira

Marion Lacoue-Nègre

Jean-François Joly

Ludovic Duponchel

Novel four dimensional approach for the structural characterization of neutral nitrogen compounds in vacuum gas oils using UHPLC-IM-QqToF analysis

The molecular analysis of complex matrices such as vacuum gas oils require powerful and very resolutive instruments such as Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS). When a structural detail about the compounds found in these cuts is targeted, ion mobility coupled to mass spectrometry (IMMS) can be used. However, the resolving power of ion mobility is not sufficient to resolve isomers from such a complex mixture. In this paper, ion mobility-mass spectrometry coupled to separative methods such as Flash-HPLC and UHPLC has been used to characterize the neutral nitrogen compounds found in vacuum gas oils. Several vacuum gas oils obtained from several industrial processes as well as different hydrotreated samples have been analyzed through a heart-cutted HPLC-UHPLC-IM-Qq-ToF analysis to target specific compounds that have been found to be problematic to hydrotreatment thanks to ESI(-)-FT-ICR MS analyses. The extraction of the macroscopic descriptors (mobility, full-width at half-maximum) allowed to extract first trends about the samples. Then, the chromatographic peaks obtained for a given alkylation degree have been divided into several retention time segments and the corresponding mobilograms have been obtained. Bi-modal distributions have been obtained and the observed CCS and MS/MS spectra suggested the presence of compact and non-compact structures. The evolution of these structures has been followed throughout hydrotreatment to identify the most intense and less reactive groups of isomers and a difference between the quantity and the reactivity of the isomers has been found. Moreover, this methodology helped to identify whether the targeted compounds were refractory to the hydrotreatment process or reactional intermediates of the hydrotreatment process.

The characterization of complex matrices such as vacuum gas oils is a major challenge in refineries. There is a real need of eco-efficient conversion processes to convert vacuum gas oils into more valuable products. In order to improve these conversion processes, two approaches can be mentioned.

The hydrotreatment (HDT) process aims at removing impurities such as sulfur, nitrogen or metals from gas oils cuts so that they can respect the legal specifications. As these are getting more and more stringent, the hydrotreatment process needs to be more and more efficient. Despite their relatively low amount among the gas oils, the nitrogen compounds are of high concern for refiners [START_REF] Sau | Effects of organic nitrogen compounds on hydrotreating and hydrocracking reactions[END_REF] . Indeed, these compounds are known to be very problematic when undergoing hydrotreatment [START_REF] Le Maître | Structural analysis of heavy oil fractions after hydrodenitrogenation by high-resolution tandem mass spectrometry and ion mobility spectrometry[END_REF][START_REF] Nguyen | Molecular level insights into straight run/coker gas oil hydrodenitrogenation by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry[END_REF][START_REF] Guillemant | Insights from Nitrogen Compounds in Gas Oils Highlighted by High-Resolution Fourier Transform Mass Spectrometry[END_REF] . Neutral nitrogen compounds are refractory while the basic ones deactivate the catalysts used and compete with sulfur compounds for their removal [START_REF] Rabarihoela-Rakotovao | Deep HDS of Diesel Fuel: Inhibiting Effect of Nitrogen Compounds on the Transformation of the Refractory 4,6-Dimethyldibenzothiophene Over a NiMoP/Al2O3 Catalyst[END_REF][START_REF] Shin | Identification and Reactivity of Nitrogen Molecular Species in Gas Oils[END_REF] . The total nitrogen content remaining in the hydrotreated samples is a key information for assessing the efficiency of the hydrotreatment. Unfortunately, it does not provide sufficient information to understand the mechanisms involved in this process [START_REF] García | In-depth modeling of gas oil hydrotreating: From feedstock reconstruction to reactor stability analysis[END_REF] . A detailed characterization of both feeds (gas oils processed by hydrotreatment) and effluents (gas oils obtained after hydrotreatment) would be very helpful to help understanding these mechanisms and thus improving the overall efficiency of hydrotreatment [START_REF] Hughey | Elemental Composition Analysis of Processed and Unprocessed Diesel Fuel by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry[END_REF][START_REF] Chen | Characterization and Comparison of Nitrogen Compounds in Hydrotreated and Untreated Shale Oil by Electrospray Ionization (ESI) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS)[END_REF] . The most used method nowadays is twodimensional comprehensive gas chromatography (GC×GC) coupled to either nitrogen chemiluminescent detector (NCD) for the absolute quantification of nitrogen compounds [START_REF] Yan | Unique selective detectors for gas chromatography: Nitrogen and sulfur chemiluminescence detectors[END_REF][START_REF] Adam | New benchmark for basic and neutral nitrogen compounds speciation in middle distillates using comprehensive two-dimensional gas chromatography[END_REF][START_REF] Chawla | Speciation of Nitrogen Compounds in Gasoline and Diesel Range Process Streams by Capillary Column Gas Chromatography with Chemiluminescence Detection[END_REF] or high resolution mass spectrometry (HRMS) mainly for the identification of nitrogen compounds [START_REF] Mühlen | Characterization of Nitrogen-Containing Compounds in Heavy Gas Oil Petroleum Fractions Using Comprehensive Two-Dimensional Gas Chromatography Coupled to Time-of-Flight Mass Spectrometry[END_REF][START_REF] Flego | N-containing species in crude oil fractions: An identification and quantification method by comprehensive twodimensional gas chromatography coupled with quadrupole mass spectrometry[END_REF] . However, information obtained with these methods is sometimes not sufficient to explain the reactivity differences between two feeds or to identify accurately the most refractory compounds. On the other hand, ultra-high resolution mass spectrometry (FT-ICR MS) provides an unsurpassed level of detail but the electrospray ionization source used to characterize nitrogen compounds undergoes important and complex ionization competition phenomena between the nitrogen families as well as possible different responses depending on the samples [START_REF] Marshall | Petroleomics: The next grand challenge for chemical analysis[END_REF][START_REF] Marshall | Petroleomics: Chemistry of the underworld[END_REF][START_REF] Ruddy | Positive Ion Electrospray Ionization Suppression in Petroleum and Complex Mixtures[END_REF] .

Thus, some approaches have already been reported in the literature combining both GC×GC quantitative features and FT-ICR MS descriptive features, but mainly for sulfur compounds and without considering both basic and neutral nitrogen compounds [START_REF] Adam | Oxidative Desulfurization Process Monitoring Using Comprehensive Two-Dimensional Gas Chromatography and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry[END_REF][START_REF] Muller | Evaluation of quantitative sulfur speciation in gas oils by Fourier transform ion cyclotron resonance mass spectrometry: Validation by comprehensive two-dimensional gas chromatography[END_REF][START_REF] Dutriez | Complementarity of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and high temperature comprehensive twodimensional gas chromatography for the characterization of resin fractions from vacuum gas oils[END_REF] . Besides, these studies were based on a small number of gas oils which is not sufficient to identify a possible matrix effect from the gas oils. In order to take into account this diesel matrix effect, the macroscopic properties of the samples can be used in multiple linear regression (MLR). This regression method has been used numerous times and is for example very useful to predict the macroscopic properties of crude oils from NMR 1 H data [START_REF] Barbosa | Application of low field NMR as an alternative technique to quantification of total acid number and sulphur content in petroleum from Brazilian reservoirs[END_REF][START_REF] Molina | Correlations between SARA fractions and physicochemical properties with 1H NMR spectra of vacuum residues from Colombian crude oils[END_REF] . Cakara et al [START_REF] Cakara | Development of a multi-variate calibration approach for quantitative analysis of oxidation resistant Mo-Si-B coatings using laser ablation inductively coupled plasma mass spectrometry[END_REF] proposed an interesting MLR approach to quantify the exact amounts of Mo, Si and B in different alloys from ICP-MS measurements. The MLR models included the possible impurities in the alloys as well as isobars species which strongly helped to correct the matrix effects observed when using ICP-MS. This method might be very helpful to quantify some complex analytes while considering the matrix effect related to the analytical method used that could prevent the quantification.

This paper aims at assessing the potential of the FT-ICR MS technique as a pseudo-quantitative tool for the analysis of nitrogen compounds in gas oils by comparing the obtained concentrations to those obtained by the reference method GC×GC-NCD. As a first step, GC×GC/HRMS was used to verify the assignments of the nitrogen compounds eluted by GC×GC-NCD and to obtain blobs depending on the DBE of the compounds. A 21 gas oils database was then analyzed by GC×GC-NCD and ESI(+/-)-FT-ICR MS but the direct comparison of both methods did not highlight clear correlation between both methods. Thus, MLR modeling was used to predict the GC×GC-NCD concentrations from FT-ICR MS measurements from the different nitrogen families in a given ionization mode.

MATERIAL AND METHODS

Vacuum gas oil samples and pre-fractionation

8 different vacuum gas oils with various industrial origins were analyzed in this study: 2 Straight Run Vacuum Gas Oils (SRVGO), 1 Heavy Coker Gas Oil (HCGO), 1 Vacuum Gas Oil from Ebullating-Bed reactor (EBVGO), 3 Hydrotreated Vacuum Gas Oils (HDT) and 1 blend (MIX, 50% SRVGO + 50% EBVGO). The macroscopic properties of these samples are shown in Table S1 in Supporting Information. All samples have been pre-fractionated to remove the hydrocarbon matrix prior to UHPLC-IM-QqToF analysis. They have been prefractionated using a Flash HPLC 2250 chromatograph (Gilson, WI, USA), following the protocol developed by Proriol et al [START_REF] Proriol | Toward New Descriptors of Basic Nitrogen Compounds in Middle Distillates by 2D NMR[END_REF] after dilution of 1g in 10 mL of heptane. 5 fractions have been obtained and evaporated using nitrogen bath (about 1 bar) without heating to prevent the evaporation of light compounds.

FT-ICR MS analysis

All samples were analyzed using a LTQ FT Ultra Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FT-ICR MS) (ThermoFisher Scientific, Bremen Germany) equipped with a 7T magnet (Oxford Instruments) and hyphenated with an ESI source (ThermoFisher Scientific) used in positive and negative modes. The chosen mass range was equal to m/z 98-1000. 70 scans with 4 µ-scans and their corresponding transients were recorded with an initial resolution set to 200,000 at m/z 300 before data processing. The ionization and ion transfer conditions for each ionization mode are described elsewhere [START_REF] Guillemant | Insights from Nitrogen Compounds in Gas Oils Highlighted by High-Resolution Fourier Transform Mass Spectrometry[END_REF] . The external mass calibration was performed using a sodium formiate clusters solution (sodium formiate provided from VWR, Fontenay-sous-Bois, France) from about 90 Da to 1000 Da. The full data processing has been described elsewhere [START_REF] Guillemant | Insights from Nitrogen Compounds in Gas Oils Highlighted by High-Resolution Fourier Transform Mass Spectrometry[END_REF] . Phase absorption and phase correction were applied on a summed transient (sum of 70 recorded transients). The corresponding mass spectrum was then noise thresholded and peak picked by an home-made software to assign the molecular formula with the following conditions: C0-50H0-100O0-3N0-3S0-3. The maximum content of heteroatoms in one molecular formula was set to 3 and 1 ppm of maximum mass error after mass recalibration based on N1[H] peaks. The relative intensities of the N1[H] compounds have been calculated as follows: the N1[H] peak intensity divided by the sum of intensities from all N1[H] peaks. The pseudo-concentrations are then obtained by multiplying the relative intensity times the amount of elemental neutral nitrogen (for ESI(-) data) or basic nitrogen (for ESI(+) data).

UHPLC-IM-QqToF analysis

The UHPLC separation was performed on a Acquity UPLC CSH Phenyl-Hexyl (Waters, Milford, MA, USA) of dimensions 100mm × 2.1 µm × 1.7Å. The oven temperature was 60°C and the flow rate was equal to 0.6 mL/min. The samples were diluted with Methanol (Optima LC/HRMS, ThermoFisher, Bremen, Germany) with a 50%/50% v/v ratio. The injection volume was 5 µL. A binary gradient was used with the aqueous mobile phase being ultra pure water with 0.5 mM of ammonium acetate and the organic mobile phase being 50% methanol-50% acetonitrile with 0.5 mM of ammonium acetate. A table describing the elution conditions is available in Table S1 in Supporting Information.

RESULTS AND DISCUSSION

FT-ICR/MS analysis

Usually, the blobs obtained by GC×GC-NCD are identified as a function of the nitrogen family (indoles, carbazoles, TetraHydroQuinolines [THQ], anilines...). However, the identification of the compounds when performing FT-ICR MS measurements is based on DBE. As an example, three differents blobs corresponding to tetrahydroquinolines, anilines and pyridines can be observed while only one family is considered for FT-ICR MS analysis. Moreover, the tetrahydrocarbazoles (THC) family is identified in a single blob while these compounds are identified in the Indoles family for FT-ICR/MS measurements as their DBE equal to 7 is contained between 6 and 8. Thus, in order to be able to compare the two methods, it was therefore necessary in a first step to create new blobs based on the DBE of the compounds rather than the nitrogen families.

To obtain these blobs, a GC×GC/HRMS analysis of the samples was required. However, GC×GC/HRMS is not very sensitive to nitrogen compounds when the whole gas oil sample is analyzed as only hydrocarbons are mostly detected. The gas oil samples were then pre-fractionated to extract the neutral and basic nitrogen compounds from the hydrocarbon matrix prior to GC×GC/HRMS analysis. The first two fractions contained the hydrocarbons. Three fractions containing the nitrogen compounds were obtained: one with the neutral nitrogen compounds (F3), one with some intermediate basic compounds (F4) and one with most of the basic compounds (F5). These fractions have been volume to volume mixed into a single solution called F3F4F5 to reconstruct the gas oil without the hydrocarbon matrix. An example of GC×GC/HRMS chromatogram obtained from the fractions of the GO 10 sample as well as their mixed solution F3F4F5 is shown in Figure 1. To facilitate the identification, the modulation periods and temperature gradients have been optimized for each fraction to maximize the chromatographic space occupancy of the fractions. The mixed solutions F3F4F5 were analyzed strictly in the same conditions as GC×GC-NCD for comparisons. The identification of the molecular formula of the compounds was possible thanks to the use of HRMS and some blobs were later created based on DBE values of these compounds.

UHPLC-IM-Qq-ToF

As a first step, the GC×GC-NCD and FT-ICR MS pseudoconcentrations obtained for the 21 gas oils have been plotted for each nitrogen family. To facilitate the reading, the most abundant compounds in the DBE families have been used, such as Indoles for the family containing the neutral DBE 6-7-8 or Acridines for the family containing the basic DBE 10-11-12. These comparisons are available in Figure 3 where the different points have been colored depending on the type of gas oil of the sample. It is quite obvious that whichever family considered, no linear trend is observed between both techniques when all samples are compared. However, if we consider the points corresponding to the same type of gas oil, Figure 3: Extraction of the EIM depending on the retention time segment considered some micro-trends could be identified. This confirms the strong need of a large and very diverse gas oil database to

CONCLUSION ASSOCIATED CONTENT Supporting Information

Figure 1 :

 1 Figure 1: Heatmap obtained for the analysis of the SRVGO sample in ESI(-) mode.

Figure 2 :

 2 Figure 2: EIC and the corresponding EIM obtained for the DBE 9 in SRVGO sample in ESI(-) mode.UHPLC-IM-Qq-ToFAs a first step, the GC×GC-NCD and FT-ICR MS pseudoconcentrations obtained for the 21 gas oils have been plotted for each nitrogen family. To facilitate the reading, the most abundant compounds in the DBE families have been used, such as Indoles for the family containing the neutral DBE 6-7-8 or Acridines for the family containing the basic DBE 10-11-12. These comparisons are available in Figure3where the different points have been colored depending on the type of gas oil of the sample. It is quite obvious that whichever family considered, no linear trend is observed between both techniques when all samples are compared. However, if we consider the points corresponding to the same type of gas oil,

Figure 4 :

 4 Figure 4: (A) Evolution of the mobility as a function of number of carbon atoms for the different DBE of all samples. (B) Evolution of the full-width at half-maximum (FWHM) as a function of number of carbon atoms for the different DBE of the feeds. (C) Evolution of the full-width at half-maximum (FWHM) as a function of number of carbon atoms for the different DBE of the hydrotreated samples.

Figure 5 :

 5 Figure 5: (A) Fragmentation spectra of C25H35N1 compound obtained for the feed and the sample HDT 3 in non-compact configuration. (B) Fragmentation spectra obtained for the feed and the sample HDT 3 in compact configuration.

Table 2 :

 2 CCS values obtained for the DBE 10 for all samples

	Sample	DBE 10, C 27 H 36 N 1 12.3-12.8 min 13.6-14.1 min
	EBVGO	212.9 Ų	201.5 Ų
	MIX	211.8 Ų	201.3 Ų
	HCGO	211.9 Ų	201.7 Ų
	SRVGO	212.2 Ų	200.6 Ų
	HDT 1	210.6 Ų	201.3 Ų
	HDT 2	212.8 Ų	200.9 Ų
	HDT 3	209.2 Ų	200.4 Ų

Comparison of the F3F4F5 solutions and whole gas oils analysis in ESI(+) mode, Comparison of the F3F4F5 solutions and whole gas oils analysis in ESI(-) mode, Characteristics of MLR models (PDF).

ACKNOWLEDGMENTS

The authors would like to thank Alexandre Verdu from Bruker for his help in data processing. The authors would also like to thank very warmly Maëva Fieu, Sophie Hénon and Romain Comte from IFP Energies nouvelles for their help with separative methods.

AUTHOR INFORMATION Corresponding Author

*alexandra.berlioz-barbier@ifpen.fr

Notes

The authors declare no competing financial interest.


Cosette Rebouissou

Séphora Sallis

Thierry Forné
email: forne@igmm.cnrs.fr

Quantitative Chromosome Conformation Capture (3C-qPCR)

Keywords: Chromosome Conformation Capture, chromatin dynamics and organization, Quantitative PCR, interaction frequency, eukaryotic genome

Many population-based methods investigating chromatin dynamics and organization in eukaryotes are based on the Chromosome Conformation Capture (3C) method. Here, we provide an updated version of the quantitative 3C (3C-qPCR) protocol for improved and simplified quantitative analyses of intra-chromosomal contacts.

Introduction

Chromosome Conformation Capture (3C) technique [START_REF] Dekker | Capturing chromosome conformation[END_REF] and derived technologies (4C, 5C, Hi-C) [START_REF] Dostie | Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements[END_REF](3)(4) have allowed researchers to explore the organization and the dynamics of the eukaryotic genomes with unprecedented resolution and accuracy [START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF][START_REF] Duan | A three-dimensional model of the yeast genome[END_REF][START_REF] Lieberman-Aiden | Comprehensive mapping of longrange interactions reveals folding principles of the human genome[END_REF][START_REF] Sexton | Three-dimensional folding and functional organization principles of the Drosophila genome[END_REF]. The creation of a 3C library is therefore a prerequisite to many 3C-based methods. By freezing all chromatin contacts present at a given time in their physiological nuclear context, and then by averaging these events over a population of several million cells, the 3C-qPCR method [START_REF] Hagège | Quantitative analysis of chromosome conformation capture assays (3C-qPCR)[END_REF] allows very accurate measurements of the relative interaction frequencies between chromatin segments, in cis, on the same chromosome (Figure 1) [START_REF] Ea | Contribution of Topological Domains and Loop Formation to 3D Chromatin Organization[END_REF]. This parameter is a key to investigate the in vivo dynamics of the chromatin because it depends not only on its fundamental biophysical parameters (compaction, rigidity, etc.) [START_REF] Court | Modulated contact frequencies at gene-rich loci support a statistical helix model for mammalian chromatin organization[END_REF] but also on important locus-specific factors which control local genomic functions (epigenetic modifications, binding of specific factors, etc.) (12,13). Here we provide an updated 3C-qPCR protocol that simplifies previously published protocols [START_REF] Hagège | Quantitative analysis of chromosome conformation capture assays (3C-qPCR)[END_REF]14,15).

Materials

1.

10% (v/v) FCS/PBS.

2.

Homogenization buffer: 2.1M sucrose, 10 mM HEPES (pH7.6), 2 mM EDTA, 15 mM KCl, 10% glycerol, 0.15mM spermine, 0.5 mM DTT, 0.5 mM PMSF, 7 µg/mL aprotinin.

31.

Genomic DNA, purified from the same species as for the 3C-qPCR experiments, and quantified by A260 measurement.

32. BAC(s), spanning the genomic locus of interest.

Methods

Cell nucleus preparation

1.

.-If working with cultured cells, proceed to step 2 below.

-If working with adherent cell cultures: trypsinize, wash and filter through 40 µm cell strainer to make a single-cell suspension before proceeding with step 2 below (see Note 5).

2.

Wash cells in PBS.

3.

Resuspend cells in 1.5 ml nucleus buffer 1.

4.

Add 0.5 ml nucleus buffer 2, mix slightly and put on ice 2 or 3 minutes.

5.

Put 1 ml in 14 ml tubes (x2 tubes) containing 4 ml nucleus buffer 3. Save a 4 µl aliquot to count nuclei in a Thoma cell (count used in step 7 below).

6.

Centrifuge 20 minutes at 11300 g at 4°C.

7.

Remove supernatant and resuspend pellets in glycerol buffer to achieve a final concentration of about 20 million nuclei in 100 µl.

8.

Transfer in a 1.5 mL tube. Freeze into liquid nitrogen and store at -80°C.

Formaldehyde crosslinking

1.

Resuspend 5x10 6 nuclei in 700 µl 3C buffer and incubate for 5 min at room temperature.

2. Add 19.7 µL 37% formaldehyde, mix by inverting the tube and incubate for 10 min at room temperature.

3.

Add 80 µL 1.25 M glycine, mix by inverting the tube and incubate for 2 min at room temperature then place on ice for 5 min.

4.

Centrifuge for 3 min at 2300 g, room temperature, remove supernatant and carefully resuspend nuclei in 1 mL 3C buffer (see Note 6).

5.

Centrifuge for 3 min at 2300 g, room temperature and remove supernatant.

Restriction digestion

1.

Resuspend nuclei in 100 µL 3C buffer and transfer to a safe-lock microtube.

2.

Add 1 L 20% SDS and incubate for 1 h at 37°C, shaking at 350 rpm on a thermomixer.

3.

Add 16.8 µL Triton mix and incubate for 1 h at 37°C, shaking at 350 rpm on a thermomixer.

4.

Take a 10 µL aliquot of the sample as the "Undigested control" (do not disturb the mixture) and store at -20°C until processing at section 3.4.

5.

Add 450 U of the selected restriction enzyme (eg. HindIII) to the remaining sample and incubate overnight at 37°C shaking at 350 rpm on a thermomixer (see Note 2).

6.

Take a 10 µL aliquot of the sample as the "Digested control" (do not disturb the mixture) and proceed with this to section 3.4. The remaining sample is processed in section 3.5.

Determination of digestion efficiency

Digestion efficiencies have a significant impact on the assays and should be carefully assessed for each restriction site involved in the analysis. Care should be taken to ensure that digestion efficiencies are in the same range for the sites of interest (see Note 2).

1.

Add 500 µL PK buffer and 1 µL 20 mg/mL proteinase K to the "Undigested" and "Digested" control samples, and incubate for 30 min at 65°C.

2.

Cool samples to 37°C, add 1 µL 1 mg/mL RNase A and incubate for 2 h at 37°C.

3.

Add 500 µL phenol/chloroform/isoamyl alcohol, mix vigorously and centrifuge for 5 min at 16,100 g, room temperature.

4.

Transfer upper aqueous layer to a new microtube. Add 50 µL 2 M sodium acetate and 1.5 mL 100% ethanol and keep at -80°C for 45 min.

5.

Centrifuge for 20 min at 16,100 g, 4°C, carefully remove supernatant and wash pellet in 500 µL 70% ethanol.

6.

Centrifuge for 4 min at 16,100 g, room temperature, remove supernatant and air-dry the pellet.

7.

Dissolve DNA in 500 µL 1X secondary digestion restriction enzyme buffer. Add 50 U secondary digestion restriction enzyme and incubate for 2 h at 37°C (see Note 7).

8.

Add 500 µL phenol/chloroform/isoamyl alcohol, mix vigorously and centrifuge for 5 min at 16,100 g, room temperature.

Ligation

1. Add 12 L 20% SDS to the digested sample and incubate for 30 min at 37°C, shaking on a thermomixer at 350 rpm.

2. Carefully transfer digested nuclei to a 12 mL tube (see Note 9). Add 3.28 mL ligation buffer and 390 µL Triton mix, and incubate for 2 h at 37°C, shaking in a thermomixer at 450 rpm.

3. Centrifuge for 1 min at 8800 g, 4°C and remove 3.27 mL supernatant to leave 500 µL in the tube. Add 6.5 µL 30 U/µL T4 DNA ligase and 3 µL 100 mM ATP and incubate overnight at 16°C, shaking on a thermomixer at 350 rpm.

DNA purification

1. Add 2 mL PK buffer, 1.5 mL nuclease-free water and 5 µL 20 mg/mL proteinase K, and incubate for 1 h at 50°C.

2.

Incubate for 4 h at 65 °C to de-crosslink the sample.

3C-qPCR 9

3. Add 4 mL phenol/chloroform/isoamyl alcohol, mix vigorously and centrifuge for 15 min at 3900 g, room temperature (see Note 10).

4.

Transfer upper aqueous layer to a new 12 mL tube and add 200 L 5 M NaCl, 1 µL glycogen and 8 mL 100% ethanol. Mix and store overnight at -20°C.

5.

Centrifuge for 45 min at 15,700 g, 4°C, remove supernatant and wash pellet in 2 mL 70% ethanol.

6.

Centrifuge for 15 min at 15,700 g, 4°C, remove supernatant, air-dry pellet and dissolve DNA in 50 µL nuclease-free water (see Note 11).

Complementary digestion

1.

Add 250 µL 2X secondary digestion restriction enzyme buffer (see Note 3) and 185 µL nuclease-free water, and transfer sample to a 1.5 mL microtube.

2.

Add 5 µL 1 mg/mL RNase A and 100 U secondary digestion restriction enzyme, and incubate for 2 h 30 min at 37°C.

3.

Add 500 µL phenol/chloroform/isoamyl alcohol, mix vigorousy and centrifuge for 5 min at 3900 g, room temperature.

4.

Transfer upper aqueous phase to a new 1.5 mL tube and add 25 µL 5 M NaCl, 1 µL glycogen and 1 mL 100% ethanol, and keep overnight at -20°C.

5.

Centrifuge for 20 min at 15,700 g, 4°C, remove supernatant and wash pellet in 200 µL 70% ethanol.

6.

Centrifuge for 4 min at 15,700 g, room temperature, air-dry pellet and dissolve DNA in 150 µL 10 mM Tris-HCl pH 7.5 (see Note 11).

Performing

4.

Repeat steps 1-2 on the new 3C stocks to precisely determine their concentration. These values will be used as the "loading controls" for final quantification (see 3.12).

Assessment of sample purity (optional)

1. Take a two-fold and a four-fold diluted aliquot of the 3C sample (section 3.7, step 6) and add genomic DNA to each aliquot to make a final total DNA concentration of 25 ng/µL.

2.

Perform qPCR for any 3C primer pair (see Note 4 and section 3.10 below) and verify that the real-time PCR quantifications are reduced according to the dilution factors. If this is not the case, then sample purity is not adequate and the DNA should either be re-purified or the sample discarded.

Real-time PCR quantifications of ligation products

PCR control template used for primer efficiency control

A control template containing all ligation products in equal amounts is used to optimise real-time quantitative PCR (qPCR) reactions and to establish the minimal amount of ligation product that can still be quantified in a reliable manner. For this qPCR control template, we recommend the use of a single BAC clone covering the genome segment under study. Alternatively, a set of minimally overlapping BAC clones mixed in equimolar amounts can be used. This BAC is then cut with the 3C restriction enzyme of choice (eg. HindIII) and religated by T4 DNA ligase. A secondary restriction enzyme (eg. StyI) can be used to linearize DNA circles which may otherwise affect primer hybridisation efficiency [START_REF] Weber | A real-time polymerase chain reaction assay for quantification of allele ratios and correction of amplification bias[END_REF] (see Note 3). It is then necessary to make serial dilutions of this reaction to obtain standard curves which cover the same range of ligation product concentrations as those that will be obtained in the 3C samples. To mimic 3C sample conditions, the final DNA concentration in these dilutions is adjusted to the amount of DNA used in the 3C samples. Thus, these dilutions are performed in a 25 ng/µL DNA solution made of genomic DNA digested with the second restriction enzyme (eg. StyI in the present protocol). Using serial dilutions of this control template, a standard curve with specific parameters (slope and intercept) is thus obtained for each of the 3C qPCR primer pairs used. These parameters will be used to correct for potential differences in primer efficiencies.

3C-qPCR data normalizationprimer efficiency and loading controls

To obtain quantification values that are corrected for potential differences in primer efficiencies, the Ct obtained for each ligation product is first normalized using the parameters of the corresponding standard curve (the slope "a" and the intercept "b" obtained in section 3.11). These values are calculated using the following formula:

Value = 10 (Ct-b)/a . For each sample, these values are then normalized to the corresponding "loading control" obtained in section 3.8. for buffer composition) using a Potter. Deposit the homogenate on a cushion of the same buffer and centrifugate at 100,000g for 40 min. Wash the pellet with wash buffer and resuspend in glycerol buffer. 9. Freeze into liquid nitrogen and store at -80°C. For muscle, the same protocol can be used except that the tissue needs to be first disrupted using an ultra-turrax before homogenization [START_REF] Milligan | H19 gene expression is up-regulated exclusively by stabilization of the RNA during muscle cell differentiation[END_REF]19).

6. Rinse the walls of the tube that have been in contact with the reaction mixture to recover all nuclei.

3C-qPCR 15 [START_REF] Lieberman-Aiden | Comprehensive mapping of longrange interactions reveals folding principles of the human genome[END_REF]. Check that the corresponding secondary digestion restriction site is absent from the PCR amplicons used to assess digestion efficiencies.

8. The efficiency of the restriction enzyme digestion should be above 60-70%, but ideally >80% is digested. Samples with lower digestion efficiencies should be discarded (also see Note 2). 9. Do not rinse the wall of the tube as this usually increase significantly the amount of undigested material. 11. If some precipitates do not resuspend, dissolve DNA by gently shaking tubes at 37°C for up to 30 min. The 3C template may be kept at -20°C for several months.

12. A260 measurements fail to provide an accurate estimate of DNA concentration in 3C samples, probably because of their limited purity. If qPCR reactions are performed in a different thermocycler (than the LightCycler, Roche) the PCR parameters may need to be optimised.

Figure caption

9 .

 9 Transfer upper aqueous layer to a new microtube. Add 25 µL 5 M NaCl and 1 mL 100% ethanol and keep overnight at -20°C. 10. Centrifuge for 20 min at 16,100 g, 4°C, remove supernatant and wash pellet in 200 µL 70% ethanol. 11. Centrifuge for 4 min at 16,100 g, room temperature, remove supernatant, and air-dry then dissolve pellet in 60 µL nuclease-free water. 12. Perform real-time PCR quantification (see Note 4 and section 3.10 below) on undigested (U) and digested (D) control samples, using primer sets that amplify across each restriction site of interest (R). To correct for any difference in the amounts of templates used in the PCR, also use primer sets to amplify control regions (C) that do not contain the restriction sites of interest. Digestion efficiency is calculated according to the following formula (see Note 8): % restriction = 100 -100 / 2^ ((CtR -CtC)D -(CtR -CtC)U)

3 . 1 .

 31 Perform qPCR reactions to obtain the Ct for each assessed ligation product on 25 ng 3C material. Set up 10 µL reaction volumes in qPCR plates: 1 µL DNA (3C, or nuclease-free water in negative control well); 7 µL nuclease-free water; 1 µL 5 µM each primer; 1 µL qPCR mix (see Note 4). Perform the following programme in a qPCR thermal cycler (see Note 12): 95°C, 10 min; 45 x: [95°C, 10 s; 69°C, 8 s; 72°C, 14 s].

10 .

 10 If the aqueous phase if very turbid after the first extraction, repeat the phenolchloroform extraction a second time.

Figure 1 :

 1 Figure 1: Principle of the Chromosome Conformation Capture (3C) assay. The

  FIGURE 1

  3. This enzyme is required to fragment the DNA which help to improve DNA accessibility and thus PCR amplification efficiencies. Please insure that this enzyme does not digest within the tested amplicons.

	4. Quantitative PCR mix composition (16): 0.24% W1 (polyoxyethylene ether
	W1); 500 µg/ml BSA; 300 µM dNTP; 50 mM KCl; 30 mM MgCl2; 1/3000 SYB R
	Green (10000X in DMSO, LONZA, ref. 50513); 16.24% Glycerol; 400 mM
	2-Amino-2-methyl-1,3-propanediol buffer to pH 8.3 using HCl; 0.4 U/µl
	platinium Taq DNA polymerase (Invitrogen 10966-034). Use a fresh glycerol
	stock, as glycerol tends to produce oxidization products that inhibit PCR
	reactions. W1 can be replaced by a combination of two detergents: 0.09% Brij
	56 (Sigma P5759) and 0.15% Brij 58P (Sigma P5884). For better
	quantification efficiencies, the usual 300 μM dNTP could be replaced by 1500
	μM CleanAmp 3'THF dNTP (Tebu Bio). We do not recommend the use of
	CleanAmp dNTP 3'TBE, as they provide similar quantification efficiencies as
	classical dNTP
	5. Nuclei preparation from tissues requires specific adaptations. For example, for
	mouse liver: homogenize the tissue in homogenization buffer (see section 2

Acknowledgements

Our work is supported by grants from the Agence Nationale de la Recherche (CHRODYT, ANR-16-CE15-0018-04), the AFM-Téléthon (grant N°21024) and the Centre National de la Recherche Scientifique (CNRS).

3C-qPCR data normalization to noise band

The data should then be normalized to compensate for experimental variations and allow comparison between different 3C-assays. For each biological sample, a Basal Interaction Level (B.I.L.) is calculated and 3C-qPCR data are normalized to this B.I.L.. We first calculate the mean interaction frequency (M) and the mean Standard Deviation (SD) of all the experimental points. Experimental points are selected if their interaction frequency (fx) is both superior to (M -SD) and inferior to (M + SD). The mean fx of the selected experimental points is corresponding to the B.I.L. to which all fx values of the experiment are normalized (14,15).

Notes

1. This buffer is intended to be compatible with the selected restriction enzyme and should thus be adapted accordingly.

Digestion efficiency in 3C experimental conditions varies largely depending on

the selected restriction enzyme. Some enzymes, like HindIII or EcoRI, have high digestion efficiencies (at least 70 to 80% is recommended for all sites investigated in a given experiment) while others, like BamHI, require sequential addition of the 450U to reach the same efficiency (add 150U for 2h, then 150U for 2h, and finally 150U overnight). Some enzyme, like SacI, does not digest at all DNA in 3C reactions. Enzymes that generate cohesive ends are recommended, as blunted ends do not allow efficient ligation.