Sunny Y Yang

David Monchaud

Dr Judy M Y Wong
email: judy.wong@ubc.ca

Global mapping of RNA G-quadruplexes (G4-RNAs) using G4RP-seq

EDITORIAL SUMMARY Guanine-rich RNAs folded into G-quadruplex structures (G4-RNAs) are captured from crosslinked RNA using a G4-specific chemical probe, BioTASQ, and mapped globally by sequencing.

PROPOSED TWEET #NewNProt for capturing RNA G-quadruplexes (G4-RNAs) using a G4-specific probe. @dmonchaud

INTRODUCTION

G-quadruplexes (G4s) are non-canonical nucleic acid structures formed by Hoogsteen bonding of four guanines to form planar guanine quartet (G-quartet) units, which π-stack on each other to form the overall columnar structure [START_REF] Burge | Quadruplex DNA: sequence, topology and structure[END_REF][START_REF] Spiegel | The Structure and Function of DNA G-Quadruplexes[END_REF] . G4s forming in DNAs (G4-DNAs) [START_REF] Rhodes | G-quadruplexes and their regulatory roles in biology[END_REF] and RNAs (G4-RNAs) [START_REF] Fay | RNA G-Quadruplexes in Biology: Principles and Molecular Mechanisms[END_REF] can affect the corresponding biological processes. G4-forming sequences are found within the genome and transcriptome of several prokaryotes, eukaryotes and viruses [START_REF] Marsico | Whole genome experimental maps of DNA G-quadruplexes in multiple species[END_REF] . Importantly, G4-forming sequences are highly saturated at sites of disease-relevance such as oncogenes, telomere, and repeat expansion regions [START_REF] Maizels | G4-associated human diseases[END_REF] . Biochemical [START_REF] Raguseo | Chemical-biology approaches to probe DNA and RNA G-quadruplex structures in the genome[END_REF] , bioinformatics [START_REF] Lombardi | A guide to computational methods for G-quadruplex prediction[END_REF] , optical imaging [START_REF] Monchaud | Quadruplex detection in human cells[END_REF] and genomic techniques [START_REF] Raguseo | Chemical-biology approaches to probe DNA and RNA G-quadruplex structures in the genome[END_REF][START_REF] Kwok | Prediction, Characterization, and Biological Application[END_REF] have been used to show that G4 formation is widespread in both the human genome and the transcriptome. However, accurately quantifying and mapping by sequencing the G4-DNA [START_REF] Biffi | Quantitative visualization of DNA Gquadruplex structures in human cells[END_REF][START_REF] Chambers | High-throughput sequencing of DNA G-quadruplex structures in the human genome[END_REF][START_REF] Hänsel-Hertsch | Genome-wide mapping of endogenous G-quadruplex DNA structures by chromatin immunoprecipitation and highthroughput sequencing[END_REF][START_REF] Zheng | Detection of genomic G-quadruplexes in living cells using a small artificial protein[END_REF] and G4-RNA sites [START_REF] Kwok | rG4-seq reveals widespread formation of G-quadruplex structures in the human transcriptome[END_REF][START_REF] Yang | Transcriptome-wide identification of transient RNA G-quadruplexes in human cells[END_REF] formed within living cells continues to be an ongoing challenge. It is important to note that G4s formation is often outcompeted by Watson-crick duplex formation, protein-binding, and G4-destabilizing helicase (G4-helicase) [START_REF] Mendoza | G-quadruplexes and helicases[END_REF] activities; thus, the G4 folding process requires windows of opportunities when the polynucleotides are single-stranded and in more openly accessible conformations. In support of this notion, it has been observed that open chromatin states are more favourable for the formation of G4-DNAs [START_REF] Hänsel-Hertsch | Genome-wide mapping of endogenous G-quadruplex DNA structures by chromatin immunoprecipitation and highthroughput sequencing[END_REF] . The high prevalence of potential G4-forming sequences in the human genome [START_REF] Lombardi | A guide to computational methods for G-quadruplex prediction[END_REF] suggests that formation of G4s likely permeates throughout most, if not all, important biological events in the flow of genetic information. G4-DNAs may play roles in various biological functions, including telomere maintenance, transcription regulation, epigenetic regulation, replication and recombination [START_REF] Varshney | The regulation and functions of DNA and RNA G-quadruplexes[END_REF] . Despite the increasing volume of research in the G4 field, much of the previous focus has been on G4-DNA rather than G4-RNA. Given the established ability of RNA strands to fold into intricate structures, which govern their functions [START_REF] Ganser | The roles of structural dynamics in the cellular functions of RNAs[END_REF] , and the superior thermostability of G4-RNA [START_REF] Fay | RNA G-Quadruplexes in Biology: Principles and Molecular Mechanisms[END_REF] , it is anticipated that G4-RNAs constitute a significant proportion of all folded G4s within living cells. G4-RNA has also been implicated in a wide range of biological processes, including translation, RNA processing, splicing, and ribonucleoprotein (RNP) formation [START_REF] Fay | RNA G-Quadruplexes in Biology: Principles and Molecular Mechanisms[END_REF] . Here, we discuss some of the current techniques used to study G4s and highlight our recently developed affinity-capture technique, called the G4-RNA-specific precipitation (G4RP) with sequencing (G4RP-seq), for studying G4-RNAs on a transcriptome-wide level.

Current methods for studying genomic and transcriptomic G4s in cells

While interest in G4s has been multiplying, there is still a limited number of techniques for studying G4s on a genome-and transcriptome-wide scale. For genome-wide studies, G4-seq [START_REF] Kwok | rG4-seq reveals widespread formation of G-quadruplex structures in the human transcriptome[END_REF] combines the polymerase-stop assay with next-generation sequencing under conditions that favour (using K+ cations or G4-stabilizing ligand pyridostatin (PDS) [START_REF] Rodriguez | A Novel Small Molecule That Alters Shelterin Integrity and Triggers a DNA-Damage Response at Telomeres[END_REF] ) or disfavour G4 formation (using Li+ cations) in purified, protein-free DNA. As folded G4s can cause polymerase-stalling, quantification of these polymerization stop sites can infer G4 folding state and generate a genomic G4 map. While the resolution is high, a major limitation of G4-seq is the in vitro experimental setting, which is not representative of the native chromatin state. Genome-wide G4 mapping by performing chromatin-immunoprecipitation sequencing (G4-ChIP-seq) [START_REF] Hänsel-Hertsch | Genome-wide mapping of endogenous G-quadruplex DNA structures by chromatin immunoprecipitation and highthroughput sequencing[END_REF] using the G4-specific antibody BG4 [START_REF] Biffi | Quantitative visualization of DNA Gquadruplex structures in human cells[END_REF] , a single-chain variable fragment antibody specific for G4, allows approximation of a more biologically relevant (i.e. "in vivo") setting. In comparison, G4seq observed over 750,000 potential G4 sites [START_REF] Chambers | High-throughput sequencing of DNA G-quadruplex structures in the human genome[END_REF] , whereas G4-ChIP-seq reported only 10,000 enriched sites [START_REF] Hänsel-Hertsch | Genome-wide mapping of endogenous G-quadruplex DNA structures by chromatin immunoprecipitation and highthroughput sequencing[END_REF] . This large discrepancy between the two techniques emphasizes the importance of the local chromatin environment in the formation of G4-DNAs.

A recently developed sequencing technique, rG4-seq [START_REF] Kwok | rG4-seq reveals widespread formation of G-quadruplex structures in the human transcriptome[END_REF] , which is conceptually similar to G4-seq, has been implemented for studying transcriptome-wide G4-RNAs. This technique exploits the effect of G4-RNAs on the processivity of reverse-transcriptase (RT) in complementary DNA synthesis. By mapping and comparing RT polymerization stop sites under G4-favourable and unfavourable ionic conditions in vitro, thousands of potential G4-RNA sites were discovered. As the query of G4 formation using purified RNA will not recapitulate the contributions of other biological factors (e.g. RNA-binding proteins, other nucleic acid structures.), rG4-seq possesses much of the same limitations as G4-seq. To that end, the RT stop-profiling method has been adapted to study G4-RNAs in vivo with an additional step of treating cells with dimethyl sulfate (DMS) before purification and RT. DMS-modified guanine bases are unable to fold into G4s in vitro and thus can be used to infer folding state in vivo indirectly [START_REF] Guo | RNA G-quadruplexes are globally unfolded in eukaryotic cells and depleted in bacteria[END_REF] . Surprisingly, DMS/RTstop profiling showed that at equilibrium, G4-RNAs are nearly all unfolded in vivo, a significant departure from observations made using live-cell imaging [START_REF] Monchaud | Quadruplex detection in human cells[END_REF]22 and in vitro quantification [START_REF] Kwok | rG4-seq reveals widespread formation of G-quadruplex structures in the human transcriptome[END_REF] methods. This apparent discrepancy is likely due to the nature of the DMS/RT-stop profiling assay, which may be more suitable for establishing the shift of G4 equilibrium rather than capturing a snapshot of the folded G4 transcriptome. Drawing inspirations from G4-ChIP-seq, we developed the G4RP-seq protocol [START_REF] Yang | Transcriptome-wide identification of transient RNA G-quadruplexes in human cells[END_REF] , which allows a broad-level snapshot view of the folded G4 transcriptome. Using G4RP-seq, we showed that G4-RNAs fold in a sequence-dependent manner and that treatment with G4-stabilizing ligands enhances these G4 folded states. Thus, the current model depicts G4-RNAs as short-lived structures which are continuously being folded and unfolded by various regulatory mechanisms inside living human cells.

These findings present us with a new perspective in which G4-RNAs are portrayed more as events rather than simple physical structures.

Development of the Biotinylated Template-Assembled Synthetic G-quartet (BioTASQ) and G4RP

Protocol

A common recurring challenge in designing G4 ligands has been the lack of specificity due to unintended off-target binding. A class of biomimetic probes, called the Template-Assembled Synthetic G-quartet (TASQ) [START_REF] Stefan | Applications of guanine quartets in nanotechnology and chemical biology[END_REF] , was developed to mitigate these off-target binding events. The chemical design of TASQ, inspired by the natural formation of G-quartets in G4s, consists of a central core with four arms, each attached to a guanine (Fig. 1a). These guanines self-assemble into a synthetic G-quartet upon association with a G4 target through end-quartet stacking. The use of the synthetic G-quartet itself as the recognition molecule provides high specificity and defined binding properties. A caveat of TASQ is its preference for targeting G4s with a parallel conformation due to the accessible end-quartets for TASQ binding without steric hindrance from the loops. This binding preference is especially useful for targeting G4-RNAs due to their parallel-conformational constraints. The extra utility of TASQ molecules is demonstrated by swapping the central core with different application-specific moieties [START_REF] Stefan | Applications of guanine quartets in nanotechnology and chemical biology[END_REF][START_REF] Haudecoeur | A Model of Smart G-Quadruplex Ligand[END_REF][25][START_REF] Laguerre | Visualization of RNA-Quadruplexes in Live Cells[END_REF] . The newest generation of TASQ molecules is a biotinylated version of TASQ (BioTASQ) [START_REF] Yang | Transcriptome-wide identification of transient RNA G-quadruplexes in human cells[END_REF][START_REF] Renard | Small-molecule affinity capture of DNA/RNA quadruplexes and their identification in vitro and in vivo through the G4RP protocol[END_REF] , allowing for affinity capture of G4 targets using streptavidin-biotin interactions. G4RP is derived from a standard RNA immunoprecipitation (RIP) protocol [START_REF] Gilbert | RNA Immunoprecipitation for Determining RNA-Protein Associations In Vivo[END_REF] ; however, instead of antibody-protein recognition in RIP, G4RP uses a ligand-nucleic acid structure recognition with BioTASQ. Like RIP and other similar methods, the G4RP protocol can be coupled with high-throughput sequencing to profile transcriptome-wide G4 distribution.

Advantages and Limitations of G4RP

Using G4 ligands as baits for G4 targets has previously been explored [START_REF] Müller | Small-molecule-mediated G-quadruplex isolation from human cells[END_REF] ; however, specificity for the intended G4 targets has been an ongoing challenge for most, if not all, G4 ligands [START_REF] Neidle | Quadruplex Nucleic Acids as Novel Therapeutic Targets[END_REF] . BioTASQ was designed with these limitations in mind by providing higher specificity due to the strictly defined binding mechanism. The use of a synthetic G-quartet as the bait provides the best-predicted interactions given that G-quartet stacking interaction is obligatory and exclusive for all G4 formations and stabilization. Some of the key advantages and limitations of G4-RNA quantification assays stem from whether the approach is direct or indirect. Direct and indirect G4-RNA identification approaches provide different perspectives due to the dynamics of G4-RNA formation, and thus the appropriate method of choice depends on the specific scientific question. Table 1 outlines some of the key differences between the two types of methods. We consider G4RP a direct approach to detect G4s due to the probe's direct association with the G4 target. However, direct approaches do have the potential pitfall of probeassisted stabilization of the G4 targets. Since BioTASQ has properties of a G4 ligand, it conceivably can stabilize G4 targets, resulting in quantification biases in capturing G4-RNAs that are not representative of their true biological states. Therefore, an in vivo crosslinking step is added prior to cell lysis and BioTASQ-assisted pulldown in G4RP, which is crucial in minimizing this bias by preventing G4-folding in vitro. In contrast, indirect approaches, including rG4-seq [START_REF] Kwok | rG4-seq reveals widespread formation of G-quadruplex structures in the human transcriptome[END_REF] and DMS/RT-stop profiling [START_REF] Guo | RNA G-quadruplexes are globally unfolded in eukaryotic cells and depleted in bacteria[END_REF] , involve nucleic acid modifications prior to subsequent screening using reverse transcriptase. Indirect approaches heavily rely on the following two assumptions: first, modifying chemicals are assumed to access all cellular RNA and modify unprotected sites uniformly within the transcriptome of living cells, which is unlikely; second, in vitro folded G4s are presumed to be the sole cause of all RT stop sites, which may lead to overestimation of the actual number of G4 sites due to the inclusion of potential polymerasestalling effects by other RNA structures [START_REF] Ganser | The roles of structural dynamics in the cellular functions of RNAs[END_REF] . Indirect approaches are likely biased against highly dynamic RNA structures as these readouts can only be estimated from highly stable structured RNAs under the set of experimental conditions [START_REF] Kubota | Progress and challenges for chemical probing of RNA structure inside living cells[END_REF] . Depending on the experimental conditions, when the labelling chemicals are usually not the limiting reagent, chemical modification in live cells can often lead to overwhelmingly modified RNAs. Thus, G4RP is considered a more appropriate method for providing a less-biased view of transiently formed G4 structures. Finally, the currently reported indirect methods do not consider the formation of intermolecular G4-RNA since the principle of RT-stop profiling is based on the effect of the cis-acting intramolecular G4s. In this sense, G4RP provides measurements that are more representative of the biologically relevant interactions due to its ability to reporting on both intraand inter-molecular folded G4-RNAs.

As indirect methods often provide high resolution data at near single base levels (e.g. chemical probing techniques, RT-stop profiling), a major limitation of G4RP is the lack of mapping resolution (>200 bases) due to the sonication step. The main challenge lies in striking a balance between over-sonication and under-sonication. Under-sonication prevents the proper release of cellular content, resulting in a reduction in resolution and lower pulldown efficiency. In contrast, over-sonication may increase mapping resolution but may also lead to disruption of the binding structure (G4 end stack) necessary for BioTASQ binding. Optimization to reach the specific goals of the experiment is highly recommended.

While sequence analysis of the BioTASQ-enriched RNA fraction in G4RP shows robust enrichment of Grich sequences, it should be noted that a minor portion of indirect interactions could be crosslinked and captured.

While G4RP does offer several advantages over the indirect polymerase-dependent methods, the choice of G4-RNA quantification method depends on the biological questions of interest. It is important to emphasize that no individual method is without limitations and caveats, and that necessary future steps should be invested in combining complementary methods in order to mitigate some of their individual limitations, thus providing a more unbiased view of the G4-RNA transcriptome.

Future applications

Theoretically, G4RP could be combined with chemical modification and in vitro RT-stop profiling to generate a high-resolution map of transcriptome-wide G4-RNA sites; however, sample processing compatibilities and assay optimization between these methods have not yet been tested. RT-stop profiling could be performed in parallel with G4RP on the same set of samples and subsequently analyzed and compared. Alternatively, RT-stop and G4RP could be performed sequentially by profiling BioTASQ-captured RNA to improve resolution. It is also yet to be determined how chemical modification (e.g. through DMS), if incorporated, could affect G4RP. While G4RP-seq could be used to infer in vivo folded state, rG4-seq (or equivalent RT-profiling methods) could be used to determine at high resolution the positions in which intramolecular G4s are more likely to form in vitro. Further optimization of the sonication step (i.e. increasing sonication duration) and/or adding ribonucleases could potentially improve resolutions of G4RP-seq by decreasing fragment sizes. However, these optimization strategies are limited to improving the resolution of intramolecular G4s. Intermolecular G4s remain a challenge to map with the currently known techniques. The use of different crosslinking agents (e.g. psoralen) with different effective distances could also be informative to elucidating a complete picture of the G4-RNA interactions. Integration of G4RP-seq with other methods for identifying RNA-RNA interactions, such as LIGR-seq [START_REF] Sharma | Global Mapping of Human RNA-RNA Interactions[END_REF] , MARIO [START_REF] Nguyen | Mapping RNA-RNA interactome and RNA structure in vivo by MARIO[END_REF] , SPLASH [START_REF] Aw | In Vivo Mapping of Eukaryotic RNA Interactomes Reveals Principles of Higher-Order Organization and Regulation[END_REF] , and PARIS [START_REF] Lu | RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure[END_REF] , may aid in identifying long-range interactions through G4s.

G4RP may also be potentially useful, when coupled with proteomics, in identifying protein partners that may interact with G4-RNAs. While the G4RP protocol has initially been optimized for human cells, it can conceivably be adapted and optimized for other model organisms, including other eukaryotes, prokaryotes and even viruses [START_REF] Marsico | Whole genome experimental maps of DNA G-quadruplexes in multiple species[END_REF][START_REF] Ruggiero | G-quadruplexes and G-quadruplex ligands: targets and tools in antiviral therapy[END_REF] . Given the unique G4 profiles of different organisms and species, G4RPseq may be valuable for studying the evolution of G4-interactome and potential inter-organism interactions (e.g. host-pathogen) [START_REF] Saranathan | G-quadruplexes: more than just a kink in microbial genomes[END_REF] .

Experimental Design

Selection of cell models and treatment endpoints. G4RP is expected to be compatible with most mammalian cell lines. The samples should be chosen with appropriate controls to ensure an unbiased comparison (e.g. vector controls for overexpression experiments). Comparisons between cell lines should include ones with similar genomic backgrounds. Typically, for drug treatments (e.g. smallmolecule compounds such as G4 ligands: BRACO-19 and RHPS4 [START_REF] Yang | Transcriptome-wide identification of transient RNA G-quadruplexes in human cells[END_REF][START_REF] Renard | Small-molecule affinity capture of DNA/RNA quadruplexes and their identification in vitro and in vivo through the G4RP protocol[END_REF] ), a dose-response profile for an observable endpoint should be generated to assess the appropriate doses for subsequent experiments.

This would ensure that the intracellular concentration of the ligand is sufficient to cause a biologically observable change in an endpoint (e.g. cytotoxicity) without leading to excessive cell death and subsequently the loss of sample. We suggest using subtoxic or low toxic dose (LD5-25; dose producing 5-25% of cell deaths). For classical G4 ligands, such as BRACO-19 and RHPS4, we have observed optimal concentrations ranging from 0.5 to 20 µM depending on the cell line. For treatments, the vehicle (i.e. solvent for compound) should be used as the control. Another factor of consideration is the duration of the treatment. Typical treatment time points include 6h, 12h, 24h, 48h and 72h. A different endpoint measure, other than cytotoxicity, may be chosen depending on the experimental goals.

Chemical crosslinking. Crosslinking could also impact sonication efficiency; however, we advise against deviating from our recommended crosslinking duration of 5 to 10 min. Most formaldehyde formulations, including those with methanol additive, are effective for crosslinking. Methanol increases cell permeabilization, which may increase crosslinking efficiency. Since formaldehyde is sensitive to light and air, we suggest storing it in aliquots in a dark and sealed environment. Fresh formaldehyde without methanol, sold in ampules, can also be considered to decrease potential variability.

Sonication and fragmentation.

Fragmentation of the RNA material is required for the G4RP. Typically, the range of fragments should fall between 200-800 nt with centering around 400-500 nt. Gel electrophoresis coupled with RNA-staining dye is a simple method for test. Other techniques requiring more advanced equipment, such as a Bioanalyzer, can also be used. We suggest using mechanical sonication as it is more consistent and sonication devices are commonly available in most institutions.

However, different sonicators (e.g. Covaris ultrasonicator, Diagenode Bioruptor, Branson Sonifier, or other equivalent equipment) will require additional optimization. We recommend testing a range of sonication durations, with the optimal total sonication duration likely to be between 2 to 4 minutes.

Different sonicators may have a recommended range in terms of the input number of cells. Please check with the manufacturer-supplied manual or recommended protocols as a guide for the initial optimization. Sonicators used for G4RP should also be equipped with a temperature control system to prevent heating and premature crosslink reversal. Mechanical sonication serves two purposes: lysing the cell and fragmenting the nucleic acids. Use of any other non-mechanical shearing alternatives (e.g.

detergent-based lysis and nuclease-mediated fragmentation) will require a different set of optimization and additional considerations of potential biases.

Bead binding. Our protocol is optimized using magnetic streptavidin-coupled beads, specifically the Promega Streptavidin MagneSphere. Other brands or types of streptavidin-coupled beads (agarose) are likely to be compatible with BioTASQ as well; however, additional optimization and validation may be necessary. BioTASQ concentration should greatly exceed the binding capacity to ensure the capture is driven by BioTASQ, not by non-specific binding to the beads. Typically, each G4RP capture uses 10-20 nmol of BioTASQ with 50-100 µg of beads. Increasing the amount of beads or switching to high-capacity beads can increase the capture yield. When altering the amounts of beads used, negative controls must be tested in parallel.

Negative control and normalization.

There are two types of controls used in G4RP. For the negative control, G4RP is performed in the same manner with biotin instead of BioTASQ. The yield of purified RNA from the negative G4RP control sample is typically much lower and serves as a control for nonspecific bindings to the beads. The negative control is necessary for early optimization but is optional after the protocol has been optimized and only negligible background is detected. The input control, a small aliquot of the material collected prior to BioTASQ binding, is critical and required for all experiments. In contrast with genomic DNA, cellular RNA content can vary widely, and this input control serves as an internal control for each G4RP experiment during the normalization step of the analyses.

PCR Primer selection.

We have included a list of recommended targets for checking the efficiency of G4RP (Table 2). However, if there are other targets of interest, additional sets of primers may be used.

These primers should undergo standard validation for specificity to the target region and PCR efficiency.

Typically, at least three biological replicates should be used to quantify each locus. For sequencing, we recommend having multiple replicates (at least 4-6). Based on the results of qPCR screening, the best 2-3 replicates can be used for library preparation. Any newly discovered region of interest from sequencing analyses should also be validated using qPCR and appropriate primers.

Library preparation and sequencing depth. For library preparation of the G4RP samples, we highly recommend the use of ribodepletion over poly-A enrichment. Due to the fragmentation step in G4RPseq, poly-A selection will likely result in loss of signal and a significant 3' bias. Always use the minimal number of PCR cycles for the library generation. In the case of low amount of input, consider pooling multiple parallel-processed samples to increase concentration. Sequencing depth will depend on the experimental question. We recommend 15-20 million uniquely mapped reads; however, this could be scaled up to 50 million reads to detect rarer events or changes. Typically, sequencing 2-3 replicates for each BioTASQ-enriched sample is sufficient for cost-effective data analysis. More replicates would improve the robustness of the statistical analysis but are unlikely to change the overall conclusions of the experiment. Sequencing of the negative control is optional and is unlikely to result in any useful data due to the low levels of the transcriptome of interest and significant rRNA background. Each condition must be accompanied by the sequencing of at least one input control. Ideally, for the most robust normalization, each BioTASQ-enriched sample can be accompanied by its respective input control.

MATERIALS Biological materials

Cell lines of interest. We have used G4RP successfully in variety of transformed or cancer-derived human cell lines of different types, including MCF7 (https://scicrunch.org/resolver/CVCL_0031), MDA MB-231 (https://scicrunch.org/resolver/CVCL_0062), HELA (https://scicrunch.org/resolver/CVCL_0030), HEK293

(https://scicrunch.org/resolver/CVCL_0045), and HT29 (https://scicrunch.org/resolver/CVCL_0320).

With proper optimization of later steps, we expect G4RP to be compatible with most organisms. Here, our protocol focuses on cultured cell lines. The use of tissue may also be compatible but will require additional optimization of tissue homogenization, crosslinking and sonication steps.

CAUTION: The cell lines used in your research should be regularly checked to ensure they are authentic and are not infected with mycoplasma.

Reagents

CRITICAL Most standard molecular and cell biology grade reagents can be obtained from various vendors and do not need to be from the ones specified here.

Cell culturing • Glycine (Fisher, cat. no. G45-212)

• 10X PBS (Fisher, cat no. BP39920)

• HEPES (Sigma, cat. no. H3375)

• NaCl (Fisher, cat. no. S271-10)

• EDTA (Sigma, cat. no. E5134)

• EGTA (Sigma, cat. no. E3889)

• KCl (Fisher, cat. no. P330)

• DTT (Fisher, cat. no. R0861)

• IPEGAL (NP40) (Sigma, cat. no. I3021)

• Tris (Fisher, cat. no. BP152)

• Chloroform (Sigma, cat. no. C2432) CAUTION: Chloroform is toxic if inhaled or swallowed. Always wear appropriate personal protective equipment and operate only inside a fume hood. All waste material should be collected separately and disposed according to institutional procedures.

• Isopropyl alcohol (Sigma, cat. no. I9516)

• Ethyl alcohol

• RiboLock RNA inhibitor (ThermoFisher, cat. no. EO0381)

• Protease-inhibitor cocktail (PIC) (optional; usually supplied as 100X; NEB #5871S)

• TRIZOL (Invitrogen, cat. no. 15596026) CAUTION: TRIZOL is a corrosive material and is toxic through ingestion, inhalation and skin contact. Always wear appropriate personal protective equipment and operate only inside a fume hood. All waste material should be collected separately and disposed according to institutional procedures.

G4RP-specific reagents

• Biotin (Sigma, cat. no. 2031)

• BioTASQ (available soon through Merck/MilliporeSigma)

• Promega Magnesphere Paramagnetic (Streptavidin beads) (Promega, cat. no. Z5481)

Reverse Transcription and PCR

• Superscript III (Invitrogen, cat. no. 18080093)

• Random hexamer pd(N)6 (Invitrogen, cat no. N8080127)

• dNTP mix (NEB, cat no. N0447L)

• SYBR green PCR-mix (BioRad, cat. no. 1725120)

Agarose Gel reagents

• UltraPure Agarose (ThermoFisher, cat. no. 16500100)

• Bleach (Clorox)

• SYBR Green II RNA Gel Stain (ThermoFisher, S7564)

Sequencing kits

Equipment

Lab equipment

• Standard cell culture incubator (e.g. HERACell, Thermos)

• Standard Benchtop centrifuge (e.g. Eppendorf)

• Temperature controlled centrifuge (or a standard unit in a refrigerated environment)

• Standard laboratory rocker

• Standard benchtop aspirator

• Cell sonicator (e.g. Covaris Ultrasonicator, Diagenode Bioruptor, Branson Sonifier)

• Tube rotator

• Nanodrop spectrophotometer (Thermos)

• Thermal cycler (Bio-Rad)

• Real-time PCR detection system (Bio-Rad)

• Gel imager (Alpha Innotech)

• Illumina HiSeq, NextSeq or NovaSeq sequencer Other consumables

• Cell and tissue culture dishes (vendor of choice)

• Cell culture treated flasks (vendor of choice)

• Cell scrapers (Corning, cat. no. 3011)

• Wide-mouth tips (VWR, cat. no. 89049)

• Covaris microtube AFA Fiber Pre-Slit Snap-Cap (Covaris, cat. no. 520045)

• PCR 8-tube strip and caps (Bio-Rad, TLS0851)

• Hard-shell 96-well PCR plate (Bio-Rad, HSP9601)

Bioinformatics Tools Prepare the stock buffer and store at 4°C for at least 1 year. A working stock should be made for every new experiment by transferring the required amount for the experiment to a conical tube and adding fresh RNase inhibitor. Use the manufacturer-recommended amount of RNAase (typically ~100 U/mL of buffer). Optionally, fresh 1X PIC may be added, especially if the downstream application involves protein.

mM Biotin

Make 1 mM stock using pure ddH 2 O. Can be stored at 4°C for at least 3 months.

mM BioTASQ

Make 1 mM stock using pure ddH 2 O. Can be stored at 4°C for at least 6 months.

Streptavidin MagneSphere Paramagnetic particles (Promega)

Always mix well before transferring beads with a pipette using wide-mouth pipette tips. The magnetic beads are stored in a stabilizing buffer (with PBS/ 0.02% wt/vol sodium azide/ 1mg/mL BSA). You may gently mix well with tapping and aliquot to small volumes (e.g. 30 µL) for later use. For a new experiment, prepare the beads by washing in 1 mL of DEPC-PBS 3 times with gentle tapping; use a magnetic rack, aspirate the buffer and resuspend in fresh DEPC-PBS to make a working stock (for one supplied 0.6 mL tube of beads at 1 mg/mL, resuspend in 100 μL of DEPC-PBS to make 6 mg/mL). While prepared beads may be stored short term (<1 week) at 4°C, it is highly recommended that fresh beads are prepared for each experiment for optimal results.

1X TAE (40 mM Tris, 20 mM acetic acid, 1mM EDTA, pH 8)

Make 50X stock and store at room temperature.

PROCEDURE

CRITICAL The G4RP protocol itself can be completed in two days. Prior to G4RP, cell culture and live-cell chemical treatment steps can take a few days to a week to set up. It is recommended that the weekly schedule is planned to avoid any interruption in the experimental pipeline. In case there are time constraints or other factors that cause anticipated interruptions, we have included a recommended pause point in which the experiments can be temporarily halted and resumed later. Please refer to Fig. 1b for an overview of the protocol.

Seeding cells and treatment with G4 ligands TIMING: 2 days to 1 week

1) Seed and treat cells as necessary in DMEM culture medium supplemented with FBS and P/S (or with the culture medium suitable for your cell line).

Generally, for most cell lines, 250,000-1,000,000 cells (minimum 150,000 cells) are seeded per 10 cm dish for a 3-4 days experiment (including seeding, cell growth and 2-3 day treatments). Seeding density can be scaled for a shorter or longer experimental period. The exact timeframes need to be optimized for different cells and treatments. Typically, the cells are harvested while in the log-phase growth with confluence at around 60-90%.

CRITICAL STEP: If G4 ligand treatment is used, make sure a suitable dose is chosen. Cell lines can respond differently to different G4 ligands. A dose-finding experiment should be performed, and a low-toxic (LD15 to LD25) dose should be chosen for subsequent experiments.

Crosslinking TIMING: 1-2 h

CAUTION: Formaldehyde is highly toxic. Always handle high concentrations (>4% formaldehyde) of formaldehyde inside the chemical fume hood. Diluted (1-4%) formaldehyde may be used outside of hood briefly (<10 min). After the crosslinking step, all formaldehyde waste should be placed in a separate waste container in the fume hood to be disposed of appropriately according to institutional procedures.

2) Make fresh 1% vol/vol formaldehyde in 1X Fixing Buffer before a new experiment.

3) Aspirate media and wash once with 10 mL of DEPC-PBS 4) Aspirate PBS and add 10 mL of 1% formaldehyde/1X Fixing Buffer to a 10 cm dish for 5 min on a rocker at room temperature. It should generally be between 5 to 10 minutes. Typically, crosslinking over 10 minutes is not recommended as over-crosslinking may limit the accessibility of epitopes.

CRITICAL STEP: After the crosslinking step, keep the sample at 4°C as much as possible.

Centrifugation should be done at 4°C as well. All buffers from this point on need to be RNase-free.

6) Remove the fixing solution, add 0.8 mL of DEPC-PBS (pre-chilled) and scrape cells into an Eppendorf tube. Keep on ice.

7) Spin tubes at 1500 rpm (200 x g) for 3 min at 4°C. 8) Aspirate supernatant (avoid disturbing the cell pellet) and wash again with 1 mL of DEPC-PBS.

Aspirate the supernatant and discard.

PAUSE POINT: You may freeze down the cell pellets at this stage and store them at -20°C to be processed later. The pellets can be stored for at least 2 months. However, for optimal results, we recommend that you do not pause at this point as processing fresh samples generally gives a better and more consistent yield for the final RNA targets from G4RP.

Sonication (TIMING: 1-2.5 h)

9) Resuspend the pellet in 400 μL G4RP buffer (supplemented with 100U/mL RNase inhibitor; it is optional to add 1XPIC). If cell pellets were frozen, thaw on ice for 30 min before resuspending in G4RP buffer. and running a 1% bleach gel [START_REF] Aranda | Bleach gel: A simple agarose gel for analyzing RNA quality[END_REF] as outlined in Mini-procedure Box (Fig. 2a).

Mini-procedure Box: RNA bleach gel 1. Make a gel mix with a final concentration of 1X TAE buffer and 1.5% Agarose in ddH2O.

2. Microwave until boiling then swirl to mix 3. Add bleach to final concentration of 1%, microwave to boil again. Swirl to mix.

4.

Pour gel mix into gel cast. Let gel cool.

5. Load RNA samples with 1X loading dye. Run at 100V for 45 min.

6. Post-stain using SYBR Green dye diluted 1:5000 in 1X TAE buffer for 30 min away from light.

Image gel on a gel imager.

? TROUBLESHOOTING 11) Spin sonicated lysate at 13200 rpm (16100 x g) for 10 min at 4°C. Move the supernatant to new tubes. The supernatant (sonicate), containing the cellular materials that are successfully released from sheared cells, will be used for the subsequent steps.

PAUSE POINT: It is possible to freeze down the sonicate for later processing. Consider using this pause point rather than the previous one if the issue with the clumpy frozen cell pellets arises.

However, for optimal results, it is recommended to continue directly to the next step. CRITICAL STEP: The amount of magnetic bead added can be optimized. The recommended amount is between 20-100 μg, depending on the brand and make of the magnetic beads. An optimization experiment using different amounts of beads is highly recommended. Generally, a higher amount increases yield but also increases non-specific background binding. Always mix well before transferring the beads to another tube.

16) Incubate for 2 h at 4°C on a rotator. 17) Use the magnet to pull beads to the side by leaving tubes on the magnet for 2 min. Shift the tubes slightly to pull beads closer together. Aspirate carefully without removing the beads.

18) Wash beads 3 times using 1 mL of G4RP buffer. Put on rotator at room temp for 5 min for each wash.

19) Wash 2 times in 1 mL of DEPC-PBS for 2 min each.

PAUSE POINT: You may store pellets in ethanol at 4°C for up to 3 days or -20°C for months.

31) Vortex briefly and centrifuge at 7500 x g for 5 min at 4°C.

CRITICAL STEP: It is unlikely that you will see a pellet here, as the amount of RNA is very low. Avoid touching the pipette tip to the bottom or creating any motion that could disturb the pellet. The addition of 25 µg of linear acrylamide during isopropanol precipitation step may also be considered to assist with recovery and visualization. ? TROUBLESHOOTING PAUSE POINT: RNA samples can be stored long term at -70°C. Freezing and thawing should be minimized to avoid RNA degradation. We recommend to immediately process the reverse transcription-qPCR to check for G4RP efficiency. The remaining stored samples may be used to process for library preparation after successful validation through RT-qPCR.

Reverse transcription and qPCR quantification TIMING: 4-5 h

CRITICAL: After adding different components, always mix by gently flicking the tube, followed by a quick pulse with a centrifuge. Do not vortex. 48) Normalization is performed by calculating the percentage of G4RP or biotin control sample against its respective input control using the following formula:

4 = 5 * (2 )
The G4RP qPCR signal is expressed as % of total amount of input going through G4RP that is effectively captured by BioTASQ. For each technical replicate, find the difference between the average of the Cq input and the individual technical replicate. Then, multiply by two to power of the previously calculated value (difference in the Cq values) by 5 (due to using 5% of the total input for normalization purposes).

? TROUBLESHOOTING Table 2. PCR primer sequences for G4RP-qPCR Gene Forward Primer Sequence (5'-3') Reverse Primer Sequence (5'-3')

NRAS ATGACTGAGTACAAACTGGTGGT CATGTATTGGTCTCTCATGGCAC VEGFA CCTTGCCTTGCTGCTCTACC AGATGTCCACCAGGGTCTCG MALAT1
AAAGCAAGGTCTCCCCACAAG GGTCTGTGCTAGATCAAAAGGCA

Ribo-depletion and Library Preparation (TIMING: 6-8 h)

49) Thaw the remaining TRIZOL-purified RNA samples (biotin-control, G4RP, and input; from step 36)

that have been validated successfully with RT-qPCR (steps 37-48) on ice.

50) It is recommended to have an additional column purification step before RNA-seq library preparation to remove any potential residual contamination (e.g. phenol, guanidium salt, etc.) and DNA contaminant. We recommend using the Qiagen RNeasy Plus Mini Kit and following the manufacturer's protocol. Elute at the low volume (<25 µL) in RNAse-free water. Quantify RNA for concentration using Qubit fluorometer.

51) For library preparation, use the Illumina TruSeq RNA Library prep kit (with Ribo-Zero) following manufacturer's protocol. We recommend a minimum RNA input of >50 ng for the library preparation process. To minimize PCR bias, higher concentration (>200 ng) is preferred if sufficient input material is available. Briefly, the steps are as follows:

• Deplete rRNA using Ribo-Zero oligos and beads.

• Thermally fragment RNA briefly at 4 min.

• Synthesize the first-strand of cDNA followed by the second cDNA strand.

• Adenylate 3' ends and ligate adapters.

• Enrich DNA fragments by PCR amplification. Use the minimum number of PCR cycles if possible (5-7 cycles).

• Quality-check and quantify library using the BioAnalyzer or equivalent methods.

CRITICAL STEP: If possible, use a higher amount (200-1000 ng) of input to improve library quality and reduce PCR cycles. G4 sequences may be sensitive to PCR by inhibiting polymerase processivity and could be underrepresented after a higher number of cycles. If the input is too low for individual samples, it is recommended to pool multiple replicate samples together.

CRITICAL STEP: The fragmentation step can be modified depending on the fragment size. Due to the sonication step earlier, the average length of RNA fragments will be reduced. To avoid over-fragmentation, the duration of the thermal fragmentation should be reduced. We recommend 2-4 min of thermal fragmentation.

52)

Pool sequencing libraries at equivalent proportion and sequence on a HiSeq, NextSeq or NovaSeq platform using pair-end and read length of 75bp or higher. We recommend the sequencing depth of each sample to be 15-20 million reads.

Sequencing Bioinformatics analyses TIMING: varies

CRITICAL An overview of the G4RP-seq analysis steps is depicted in Fig. 2b. For investigators who are less experienced with bioinformatics or lack a local workstation computer (minimum requirement: >8 core processor, >32 GB memory, >3 TB hard drive) performing data analysis through a remote Galaxy server [START_REF] Goecks | Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences[END_REF] (usegalaxy.org), which contains all the required bioinformatics packages, is highly recommended.

53) Quality-check raw FASTQ file using FASTQC and trim reads using Trimmomatic (38) (or equivalent read-trimming tool) with default settings.

54) Align reads to the target genome (e.g. human genome hg19 or hg38 assembly) using HISAT2 (39) using default settings. The output file should be formatted as the compressed binary version of sequence alignment files (BAM format). Integrative Genome Viewer (IGV) (40) can then be used to visualize these alignments. To load in IGV, index files will need to be generated from igvtools for each BAM file.

55) Prepare and download a gene annotation file (in GTF format) from UCSC. This can be accessed at http://genome.ucsc.edu/cgi-bin/hgTables . Make sure to choose the right genome and assembly version. Choose "Genes and Gene Predictions" for the group and "NCBI RefSeq" in the track. To use gene symbols as an attribute of interest, choose "refFlat" for the table. The parameters may be changed depending on what attributes you want to be listed. Select "genome" for region and GTF as output format. The resulting GTF file can be either downloaded directly or sent as output to Galaxy.

56) Count features and annotate the aligned reads using HTSeq-count (41) with the prepared GTF file.

Use "Union" mode and set values as follows: feature type=exon, ID attribute=gene_id. Depending on the library prep kit used, you can set the strandedness mode either "stranded" or "nonstranded." The "stranded" setting would keep data on the directionality of the transcript. Step 22-36, RNA purification: 2 hours

Step 37-48, Reverse transcription and qPCR quantification: 4-5 hours

Step 49-51, Ribodepletion and library preparation: 6-8 hours

Step 52-60, Sequencing and bioinformatics -varies (depending on sequencing queues and computational power)

TROUBLESHOOTING

Please refer to Table 3 for advice on troubleshooting.

ANTICIPATED RESULTS

Sonication check

Due to the different sonication techniques used by different laboratory groups, it is advised that a sonication check be performed using different sonication conditions. For example, for the Covaris m220

Ultrasonicator, we divided a crosslinked sample and performed parallel comparisons between different sonication conditions (sonication duration: 0 min, 2 min and 4 min) under the same settings. Sonicated samples can be heated for 1h to reverse crosslinks, and the RNA can be extracted using TRIZOl or RNA column purifications. The purified RNA can then be observed on an RNA bleach gel stained with SYBR green nucleic acid gel stain [START_REF] Aranda | Bleach gel: A simple agarose gel for analyzing RNA quality[END_REF] . As depicted in Fig. 2a, absence of sonication would result in an inefficient release of RNA from crosslinked material. Note that crosslink reversal is inefficient in unsonicated material and thus would result in a loss of extracted RNA, as shown by the lack of RNA band on the gel for the 0 min condition. Generally, a sonication duration of 2-4 min is sufficient, yielding a range of bands between 200-800 nt. Longer sonication duration leads to a shorter average length of RNA fragments, as seen by the shifting of the median band length between the 2 and 4 minutes of sonication conditions. It is important to note that, while longer sonication may help increase resolution, it may also disrupt some interactions. Thus, a balanced approach is required to prevent under-or over-sonication.

RNA yield from G4RP

After TRIZOL-purification, the purified RNA can be quantified using Nanodrop or other comparable methods. A lower A260/280 ratio (<1.8) is usually indicative of contamination from TRIZOL extraction.

However, A260/280 value may not be reliable if the sample concentration is too low. In that case, a more sensitive method, such as Qubit, may be employed. Typically, the concentration of the 5% input sample is around 100 ng/μL (with a 30 μL yield = 3 μg). The concentration of the BioTASQ-enriched sample varies around 100-200 ng/μL (30 μL yield of 3-6 μg). The concentration of the biotin control sample is much lower at ~50 ng/μL (30 μL yield of 1.5 μg), as the biotin control consists mostly of nonspecific rRNA binding to the beads. Regardless of purity or concentration, we highly recommend column purification before further processing (i.e. ribodepletion and library preparation) for RNA-seq.

G4RP-qPCR quantification

RT-qPCR quantification of the G4RP samples typically yields high signal-to-background ratios (>20 fold enrichment) when comparing % input yield from BioTASQ versus the biotin control sample (Fig. 2b).

Ligand-induced change ranges from 2-fold to as high as 12-fold, depending on the RNA target and G4 ligand used.

G4RP-seq analysis

After processing the raw sequencing files through a series of bioinformatics tools (Fig. 3a), the differential expression values (output from DESeq2) derived from comparing the BioTASQ sample with its corresponding input control, are used to determine the BioTASQ enrichment score. A quick indicator of a successful G4RP-seq is to rank the transcripts by enrichment score and check for an increase in GC content. At baseline levels (derived from samples not treated with a G4 ligand), the enrichment score is expected to be positively correlated with the GC% of the gene transcript (Fig. 3b). Signals of aligned reads can be visualized using IGV (Fig. 3c). The biotin control is expected to show negligible levels with reads primarily aligned to rRNA regions. By comparing the BioTASQ signal with that of the input control, the relative level of BioTASQ interaction with the target can be elucidated (Fig. 3c). An example of a high G4-ligand-inducible target is the long non-coding RNA, MALAT1. The raw signals show much higher alignments in the G4-ligand-treated samples compared to that of the untreated sample.

5 )

 5 Quench crosslinking by adding glycine to a final concentration of 125 mM and incubate 5 min on a rocker at room temp.CRITICAL STEP: The duration of the formaldehyde crosslinking step is important. Try to keep it as consistent as possible between samples. Stagger the addition of formaldehyde and quenching if processing several samples at the same time. You may want to optimize the duration of crosslinking.

? TROUBLESHOOTING 10 )

 10 Sonicate on a Covaris m220 focused ultrasonicator using 10% duty factor "Chromatin Shearing" preset for 2 min at 7°C temperature setpoint. The Covaris AFA Fiber microtube-130 is used for the sonication. Mix well before adding the resuspended cells to the tube. Make sure there are no bubbles when adding the sample to the AFA microtubes prior to sonication. Each sonication tube should contain exactly 130 μL corresponding to approximately 2-3 million cells. Repeat until all the resuspended cells are sonicated.CRITICAL STEP: Optimization with specific sonicators (Covaris, Diagenode, and other brands) is required. The sonication protocol should be similar to that recommended for ChIP according to sonicator-specific published protocols. We recommend that different durations of sonication be tested. Generally, the sonication duration should not exceed that recommended for ChIP, and a good starting point for optimizing the duration is to use half of what is recommended for ChIP. Make sure the temperature is kept around 2-7°C. Sonication can be checked by proceeding toSteps 22-36

Binding and washes TIMING: O/N + 4 h 12 )

 12 Each sonicate of ~390 μL can be aliquoted into two tubes (each with 180 μL): one for the biotin control and the other for BioTASQ. If the volume is insufficient (<360 μL), the volume can be adjusted by adding the small amount of the remaining G4RP buffer, prepared previously, to a final volume of 390 μL. Save 8.75 μL of the remaining sonicate as a 5% input control. The input control can be stored temporarily at 4°C then processed later with the other samples. 13) Add 20 μL of 1 mM stock biotin (20 nmol) to a final concentration of 100 μM to the control tube.Add 20 μL of 1 mM stock BioTASQ (20 nmol) to a final concentration of 100 μM to the G4RP tube. 14) Incubate overnight at 4°C on a rotator. 15) Next day, use wide-mouth tips to add 10 μL of MagneSphere magnetics beads (60 μg) to each tube.

32 )

 32 Slowly remove the ethanol leaving approximately 10 μL of liquid at the bottom of the tube. 33) Keep the lids of the tubes open and air dry for 10 min at room temp. 34) Add 30 μL of RNAse-free water and pipette up and down. 35) Incubate at 55°C for 15 min. 36) Check RNA concentration and quality using Nanodrop.

37 ) 39 ) 42 ) 45 )

 37394245 Thaw purified RNA (including all G4RP, biotin control, and input control samples) from Step 36 on ice.38) A master mix can be made with pdN6 and dNTP. Add 2 μL to each tube containing RNA and mix: Incubate at 65°C for 5 min. 40) Place on ice for 5 min. 41) Make a master mix of the following components and gently mix by flicking. Add 20 μL to each tube: Gently mix by flicking the RT reaction tube and pulse spin by brief centrifugation.43) Incubate at room temp for 5 min.44)Then, incubate at 55°C for 45 min. These incubations (steps 43-45) can be performed on a thermocycler. Inactivate reaction by incubating at 70°C for 15 min and cool down to room temp. Proceed to realtime qPCR with the appropriate primer set and SYBR mix of your choice. Table2includes primer sets for several genes that have been tested in MCF7, MDA MB-231 and HEK293 cell lines. However, depending on your experimental objectives and models, you may require a different set of controls. PAUSE POINT: The cDNA samples can be stored at -20°C long term. 46) Make a master mix with the appropriate primer set. Typically, we suggest the use of two to three technical replicates. Use qPCR plates or strip tubes compatible with the real-time qPCR machine. An example of a single qPCR reaction is as follows: the real-time qPCR using the default amplification protocol for your instrument. It should typically take around 20-30 cycles to reach exponential phase (~2-3 hours).

57)TIMINGStep 1 ,

 1 Use the raw count output from HTSeq-count as input for DESeq2(42) for normalization and differential expression analysis.58) RunDESeq2 for the BioTASQ sample vs input control for each treatment condition to generate relative BioTASQ enrichment scores (ES), which are log fold change readout between the test conditions for each transcript. The gene transcripts can be ranked according to their enrichment score. It is important to note that all subsequent comparisons are relative measures. 59) Compare the enrichment scores between conditions to quantify ligand-induced changes in G4 RNA structures. The enrichment score change (ΔES) is calculated as follows: ES (treated) -ES (untreated). 60) Using Excel, filter transcripts by counts (as a measure of RNA abundance) and fold change. The filter threshold can differ depending on the quality and depth of sequencing. Cell culture phase (including seeding and treatment): 2 days to 1 week (depending on cell culturing experimental setup) Steps 2-8, Crosslink: 1-2 hours (depending on the number of samples) Steps 9-11, Sonication: 1-2.5 hours (depending on the number of samples) Steps 12-21, Binding and washing: overnight + 4 hours

Figure 1 .

 1 Figure 1. Overview of G4RP. a, Chemical structure of BioTASQ in its open (in absence of a G4 target) and

Figure 2 .

 2 Figure 2. Expected results from G4RP. a, An example of an bleach RNA gel (1.5% agarose w/v, 1%

Figure 3 .

 3 Figure 3. Expected results from G4RP-seq. a, Schematic of the G4RP-seq bioinformatics analyses. b,

Table 1 . Indirect vs direct methods in studying transcriptomic G4-RNAs Indirect method (rG4-seq, DMS/RT-profiling) Direct method (G4RP-seq)

 1

	Readout method	Reverse-transcription stalling +	Probe-binding/extraction +
		sequencing	sequencing
	Methods to capture	Modification of nucleic acids in live	Chemical crosslinking
	biological state	cells
	Resolution	High	Low
	Targets	-Intramolecular G4s	-Intramolecular G4s
			-Intermolecular G4s
	Enrichment method	Poly (A) enrichment	Ribo-depletion
	Biases/ Limitations	-in vitro biases	-Probe-assisted stabilization could
		-DMS modification could lead to	lead to overestimation of G4
		underestimation of dynamic	structures
		structures	-a small portion of crosslinked indirect
		-Overestimation of G4 structures	interactions could be captured
		by counting stops induced by other
		structures
		-Not able to read 5' G4 if there is a
		strong RT stop caused by 3' G4
		signal

  DESeq2 42 (https://scicrunch.org/resolver/SCR_015687)https://doi.org/doi:10.18129/B9.bioc.DESeq2

	Reagent Setup
	CRITICAL: All buffers should be sterile and carefully prepared using RNase-free water.
	DEPC H 2 O
	0.1% vol/vol DEPC-treated water can be made by adding 1 mL DEPC to 1000 mL of ddH 2 O. Mix
	and incubate at room temperature (20-22°C) for 1h before autoclaving.
	• Trimmomatic 38 -https://github.com/timflutre/trimmomatic
	• HISAT2 39 (https://scicrunch.org/resolver/SCR_015530) -http://daehwankimlab.github.io/hisat2/
	• Integrative Genomics Viewer 40 (https://scicrunch.org/resolver/SCR_011793) -
	http://software.broadinstitute.org/software/igv/

• HTSeq-count

[START_REF] Anders | HTSeq--a Python framework to work with high-throughput sequencing data[END_REF]

(https://scicrunch.org/resolver/SCR_005514)https://htseq.readthedocs.io/en/master/ • DEPC-PBS Dilute 10X PBS (RNase-free) to 1X in DEPC H 2 O or nuclease-free water from a vendor. Store at room temperature. 1X Fixing buffer (50 mM HEPES KOH pH 7.5, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0 in DEPC H 2 O) Prepare a 5X stock solution and store at room temperature for at least 1 year. 1M Glycine Prepare in DEPC H 2 O and filter sterilize. Store at 4°C for up to 2 months. G4RP Buffer (150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT, 0.5% vol/vol NP40 in DEPC H 2 O or nuclease-free water)

Table 3 . Troubleshooting table

 3

	Step Problem	Possible Reason	Solution
	9	Cells are too clumpy and difficult to resuspend	Freezing after formaldehyde may cause clumping	Do not freeze down pellet and directly proceed to the sonication step.
		No bands
		corresponding to
		purified RNA	Crosslink reversal is	Increase duration of the crosslinking
		from sonication	insufficient	reversal step to 2h.
		check fraction
		visible on gel
	10	Either faint bands or bands with apparently high	Cells not sonicated properly.	Reoptimize the sonication step. Increase sonication duration.
		molecular weight
		observed in
		purified RNA of sonication check fraction	Cellular components not released into the supernatant	Add 0.1% (wt/vol) SDS to resuspended cells before sonication
				Wash twice, instead of once, with ethanol
			Contamination from	during the TRIZOL extraction. This will
	36	Low A260/280 ratio for RNA	TRIZOL components (e.g. Phenol), protein	normally not impact qPCR quantification. Perform an additional column purification
			or DNA.	on RNA before sequencing library
				preparation.

ACKNOWLEDGEMENTS

This work is supported by the Research Reinvestment Funds from the University of British Columbia and Agence Nationale de la Recherche (ANR-17-CE17-0010-01). Pauline Lejault is warmly acknowledged for her critical analysis of the G4RP protocol and of the manuscript.

DATA AVAILABILITY

All relevant data are available upon request. The G4RP-qPCR dataset (Fig. 2) is publicly available on FigShare (https://doi.org/10.6084/m9.figshare.16684870). The raw sequencing data used in the examples shown in Fig. 3 in this protocol are publicly available and accessible at GSE112898.

COMPETING INTERESTS

The authors declare no competing interests.