Angela Schwarm
email: schwarm@fbn-dummerstorf.de

Sylvia Albrecht

Sylvia Ortmann

Christian Wolf

Marcus Clauss

Digesta retention time in roe deer Capreolus capreolus, as measured with cerium-, lanthanum-and chromium-mordanted fibre

Keywords: Ruminant, Browser, Mean retention time, Passage rate, Marker

Studies on particle retention time in ruminants are commonly conducted utilizing chromium (Cr) and/or a lanthanide bound to feeds. Both types of markers have different chemical properties which potentially bias estimates of digesta retention-their combined use is therefore a topic of ongoing discussion. In order to evaluate the suitability of two lanthanides for studies assessing the passage kinetics of different-sized particles, we measured the mean retention time in roe deer with cerium (Ce) and lanthanum (La) labelled to the same particle length and compared it to Cr-mordanted fibre of the same size. We expected a simultaneous excretion of Ce-and La-labelled fibre, but a delayed excretion of Crmordanted fibre compared to the other markers. In this study, the mean retention times of Ce-and La-labelled fibre did not differ significantly, and Cr-mordanted fibre was retained on average 5 h longer than Ce and La. Despite the limitation of the small sample size, Ce and La showed the same excretion pattern and can therefore be recommended as mordants in studies assessing passage kinetics of different particle sizes.

Introduction

Studies on particle retention time in ruminants are commonly conducted utilizing chromium (Cr) or lanthanides bound to feeds. The lanthanide series consists of 15 elements with similar chemical properties, with atomic numbers 57 through 71, from lanthanum (La) to lutetium [START_REF] Bernard | Use of rare earth elements as external markers for mean retention time measurements in ruminants[END_REF]. Cr is the most widely used marker in nutrition studies and has a better reputation than lanthanides like ytterbium (Yb) and cerium (Ce), which have been criticized to potentially dissociate from labelled particles [START_REF] Beauchemin | Evaluation of markers, sampling sites and models for estimating rates of passage of silage or hay in dairy cows[END_REF][START_REF] Combs | Retention of rare earths by hay particles following incubation in fresh or autoclaved rumen fluid[END_REF][START_REF] Huhtanen | Comparison of methods, markers, sampling sites and models for estimating digesta passage kinetics in cattle fed at two levels of intake[END_REF]; this property has been offered as an explanation for the observed shorter (7-29 h) mean retention time (MRT) of Yb-labelled fibre compared to Cr-mordanted fibre of the same size (Table 1). On the other hand, it has been demonstrated that Cr largely decreases cell wall digestibility [START_REF] Udén | Investigation of chromium, cerium and cobalt as markers in digesta rate of passage studies[END_REF], which in turn could lead to the observed longer retention time compared to a Yblabelled fibre of the same size [START_REF] Beauchemin | Evaluation of markers, sampling sites and models for estimating rates of passage of silage or hay in dairy cows[END_REF][START_REF] Huhtanen | Comparison of methods, markers, sampling sites and models for estimating digesta passage kinetics in cattle fed at two levels of intake[END_REF]; Table 1). As a consequence, in studies which aim to assess excretion kinetics of large vs. small particles in ruminants, the combined use of Cr and the lanthanide Yb as mordants is not the optimal choice. Looking at studies with cattle fed different-sized particle markers, using Cr and lanthanides as Ce or La, the observed difference in their respective MRT was greater when Cr was bound to the large particles [START_REF] Lechner-Doll | Seasonal changes of ingesta mean retention time and forestomach fluid volume in indigenous camels, cattle, sheep and goats grazing a thornbush savannah pasture in Kenya[END_REF]) rather than to the small particles [START_REF] Schwarm | Excretion patterns of fluid and different sized particle passage markers in banteng (Bos javanicus) and pygmy hippopotamus (Hexaprotodon liberiensis): two functionally different foregut fermenters[END_REF][START_REF] Lechner | Differential passage of fluids and different-sized particles in fistulated oxen (Bos primigenius f. taurus), muskoxen (Ovibos moschatus), reindeer (Rangifer tarandus) and moose (Alces alces): rumen particle size discrimination[END_REF]. From this, we concluded that it would be probably advantageous to use two lanthanides bound to different particle lengths rather than the application of Cr and a lanthanide in digesta passage studies in ruminants. So far, only two studies evaluated the combined use of lanthanides, namely Yb combined with dysprosium (Dy) or Ce; these lanthanides were excreted simultaneously from the digestive tract of cattle and rabbit if bound to the same particle length [START_REF] Goetsch | Ruthenium phenantrholine, dysprosium and ytterbium as particulate markers in beef steers fed an all-alfalfa hay diet[END_REF][START_REF] Gidenne | Rate of passage in the rabbit digestive tract: influence of marker dosing time, ileal cannulation and marker type[END_REF]; Table 1). The objective of our trial, as part of a larger study, was to assess the MRT with two lanthanides labelled to the same particle length in roe deer. We chose Ce and La as markers, and a synchronous excretion was expected. Additionally, we intended to feed Cr-mordanted fibre of identical size and expected a delayed excretion compared to the other markers. However, due to the experimental design of the larger study, Cr marker had to be fed 1 day prior to the application of the lanthanide markers. Because food intake was relatively constant throughout the trial, we decided to show and discuss the results nonetheless. Finally, the obtained data are compared to the data of particle retention time in roe deer published so far [START_REF] Holand | Seasonal dynamics of digestion in relation to diet quality and intake in European roe deer (Capreolus capreolus)[END_REF][START_REF] Van Wieren | Digestive strategies of ruminants and nonruminants[END_REF][START_REF] Behrend | Seasonal faecal excretion, gut fill, liquid and particle marker retention in mouflon Ovis ammon musimon, and a comparison with roe deer Capreolus capreolus[END_REF]).

Materials and methods

The trial, as part of a larger study, was performed with four adult roe deer (17-24 kg) at the field research station of the IZW in June 2006. Details of the animals are recorded in Table 2. Animals 2, 3 and 4 were born in summer 2004 at the field research station. Animal 1 was bought in 1999; its birth date is unknown. Animals were usually kept in groups in meadow enclosures, but were moved to individual pens 4 weeks before the trial started. On this occasion, animals were weighed. Individual pens measured 25-30 m 2 and had a carpeted floor. Individuals were offered on average 440 g dry matter per day of a pelleted compound feed for dairy calves (Märka KA-18-3, Märkische Kraftfutter GmbH, Guben, Germany) and 68 g dry matter per day pelleted meadow hay (Pre Alpin Compact, Agrobs GmbH, Münsing, Germany). The chemical composition of the pellets for dairy calves was 23% crude protein (CP), 17% neutral detergent fibre (NDF) and 7% acid detergent fibre (ADF) on a dry matter basis. The pelleted hay contained 15% CP, 50% NDF and 29% ADF on a dry matter basis. Animals had food access from approximately 9A.M. to 9P.M. A fifth animal refused to eat and was excluded from the trial. Respective food items offered and leftovers were quantified on a daily basis by weighing. Drinking water was always provided ad libitum. Digesta MRT through the gastrointestinal tract (GIT) was determined with 2-mm particles either labelled with Ce, La or Cr. Markers were prepared according to [START_REF] Udén | Investigation of chromium, cerium and cobalt as markers in digesta rate of passage studies[END_REF] and [START_REF] Heller | Retention of fluid and particles in the digestive tract of the Llama (Lama guanacoe f. glama)[END_REF]. Preparation and analytical procedures are described in detail elsewhere [START_REF] Schwarm | Excretion patterns of fluid and different sized particle passage markers in banteng (Bos javanicus) and pygmy hippopotamus (Hexaprotodon liberiensis): two functionally different foregut fermenters[END_REF]. In brief, dried grass hay was ground through a 2-mm 2 perforated screen with a Retsch mill (SM 2000, Retsch GmbH & Co. KG, Haan, Germany). Dust and smaller particles were removed by dry sieving. After neutral detergent treatment, 100 g dried particles each were incubated either with 54 g lanthanum [III] chloride hydrate (LaCl 3 ⋅H 2 O), with 75 g cerium(III) chloride heptahydrate (CeCl 3 ⋅7H 2 O) or with 33 g sodium dichromate dihydrate (Na 2 Cr 2 O 7 ⋅2H 2 O). After mordanting at 37°C (Ce, La) or 100°C (Cr) and thorough washing with tab water, the fibre was dried at 65°C. We applied 1.5 g of each marker per animal. As outlined in "Introduction", only the Ce and La markers could be fed simultaneously due to the experimental design of the larger study. Ce-and La-labelled fibres were mixed with 15 g ground compound feed for dairy calves, pressed to pellets and fed in the morning of trial day 1 (June 11) prior to the daily ration.

The average time between start (6A.M.) and end (9A.M.) of marker feeding was taken as the time of marker uptake. Faeces were collected in toto the following 5 days (June 12-16). Cr marker had to be fed 1 day prior to the application of the other markers. At day 0 of the trial (June 10), Cr-mordanted fibre (with 15 g concentrate compound) was offered at 6A.M. The pens were checked at 7A.M., 9A. M. and 12A.M. for marker uptake, after which the daily ration was fed. Marker uptake in animals 1 and 2 was between 9A.M. and 12A.M. (the marker was not touched by these animals before 9A.M.). Animals 3 and 4 totally consumed the marker between 6A.M. and 7A.M.. The middle of the recorded time period was used as the starting time for MRT calculations (10:30A.M. in animals 1 and 2 and 6:30A.M. in animals 3 and 4). Faeces were collected in toto the following 6 days (June 11-16). Individual enclosures were checked for defaecations seven times per day on days 1 to 5 of the trial (1:30A.M., 6A.M., 9A.M., 12A.M., 3P.M., 6P.M. and 9P.M.) and two times at day 6 of the trial (9A.M., 9P.M.). The average time between collections was taken as the time of defaecation. Individual defaecations were cleaned from hair, weighed, and the whole sample or an aliquot (after thoroughly mixing; max. 300 g) was stored frozen until drying at 60°C. Dry matter (DM) content of food and faecal subsamples was determined by drying an aliquot at 103°C to constant weight. Ground faecal samples were microwave-digested and analysed for Ce, La and Cr Marker concentrations adjusted for visual comparability. Note: Ce and La marker were ingested simultaneously as a pulse dose within 3 h. Cr marker uptake was 25 h prior to Ce and La; it was ingested within 1 h. The animal had a dry matter intake during the 6 days of the trial of 46± 2 g kg -0.75 day -1 . Intake the day prior to Cr marker feeding was 42 g kg -0.75 day -1 Source: 1 Behrend (1999); [START_REF] Behrend | Seasonal faecal excretion, gut fill, liquid and particle marker retention in mouflon Ovis ammon musimon, and a comparison with roe deer Capreolus capreolus[END_REF]), 2 Holand (1994), 3 Van Wieren (1996) a MRT was measured after offering a single amount of 10% of the diet stained with basic fuchsin at the start of the measuring period during 4 h. Faecal grab samples were taken concentration by inductively coupled plasma mass spectroscopy (ELAN 6,000, PerkinElmer Life and Analytical Sciences, Milano, Italy) following [START_REF] Schwarm | Excretion patterns of fluid and different sized particle passage markers in banteng (Bos javanicus) and pygmy hippopotamus (Hexaprotodon liberiensis): two functionally different foregut fermenters[END_REF].

The MRT in the total GIT was calculated according to [START_REF] Thielemans | Mesure du transit gastrointestinal chez le porc a l'aide des radiolanthanides. Comparaison avec le mouton[END_REF]

Results

Without exception, the resulting marker excretion curves for the simultaneous fed Ce-and La-labelled particles (both 2 mm) show a simultaneous processing of the two mordants (Fig. 1). Accordingly, the MRT for Ce-and La-labelled fibres did not differ significantly (paired t test, n=4; mean ± SD, 37±6 h vs. 38±7 h, p=0.391). Individual MRT measurements, as well as the dry matter intake (DMI), are recorded in Table 2.

The excretion curve for Cr deviated from the Ce and La excretion curves (Fig. 1). Cr-mordanted fibre was retained on average 5 h longer than Ce and La (MRT, 43±9 h; Table 2) despite the same particle size of 2 mm; however, this difference was not significant (GLM RM, p=0.383). Only in one animal (no. 3) were Cr-mordanted particles excreted as fast as Ce-and La-labelled particles. In this animal, food intake was not constant over time (Table 2), being lower the day prior to Cr marker feeding compared to the subsequent days.

The relative DMI (mean ± SD) in roe deer was 48±5 g (food) kilogram (body mass) -0.75 per day (Table 2).

Discussion

Lanthanides

Despite the limitation of the small sample size, the results of this study support the hypothesis of a simultaneous excretion of the lanthanide markers Ce and La labelled to the same-sized particles from the ruminant digestive tract. This result is in accordance with a study using two other lanthanides, namely Dy vs. Yb, bound to same-sized particles as passage markers in cattle [START_REF] Goetsch | Ruthenium phenantrholine, dysprosium and ytterbium as particulate markers in beef steers fed an all-alfalfa hay diet[END_REF]. Also in a study with rabbits, same-sized particles labelled either with the lanthanide Ce or the lanthanide Yb were excreted simultaneously [START_REF] Gidenne | Rate of passage in the rabbit digestive tract: influence of marker dosing time, ileal cannulation and marker type[END_REF]; Table 1). Since we did not find a systematic difference in the excretion of Ce-and La-labelled fibres of the same length, conclusions on animal physiology as those of [START_REF] Schwarm | No distinct difference in the excretion of large particles of varying size in a wild ruminant, the banteng (Bos javanicus)[END_REF] and [START_REF] Lechner | Differential passage of fluids and different-sized particles in fistulated oxen (Bos primigenius f. taurus), muskoxen (Ovibos moschatus), reindeer (Rangifer tarandus) and moose (Alces alces): rumen particle size discrimination[END_REF] based on lanthanide marker excretion of different lengths can be supported by our study. These authors found in different ruminants that particle passage kinetics and retention of 10-mm particles labelled with Ce and 20-mm particles labelled with La did not differ. They concluded that particle retention in the ruminant forestomach is not a continuous function of particle size (i.e. the larger a particle, the longer it is retained) but rather threshold-dependent (i.e. all particles above a certain size, e.g. 10 mm, are retained for the same period of time).

Chromium vs. lanthanides

Conclusions concerning Cr marker excretion are limited by the fact that Cr-mordanted fibre could not be fed simultaneously with Ce and La, but was ingested on average 21 h (animals 1 and 2) and 25 h (animals 3 and 4) prior to Ceand La-labelled fibres. MRT of Cr-mordanted fibre did not differ significantly from fibres labelled with lanthanides (Ce, La), which may be due to the low sample size and the fact that in one animal (no. 3) relative DMI prior to Cr marker feeding was 50% lower than the average intake of the following 6 days (Table 2). The animals with a relatively constant intake (nos. 1, 2 and 4) excreted Crmordanted fibre 4-9 h after the Ce-and La-labelled fibres (Table 2). It seems that irrespective of the type of lanthanide used (Yb, see data collection in Table 1; Ce/La, this study), Cr has a comparably longer MRT. Therefore, results from studies where small, Cr-mordanted fibres were shown to be Fig. 2 Relationship between relative dry matter intake (g kg -0.75 day -1 ) and mean retention time (h) of feed particles in the gastrointestinal tract of roe deer fed either roughage (solid line) or concentrates (dashed line). Spearman correlation coefficient (SCC): roughage: N=8, SCC=-0.904, p=0.002; concentrates: N=8, SCC=0.419, p=0.301. Source: roughage: [START_REF] Holand | Seasonal dynamics of digestion in relation to diet quality and intake in European roe deer (Capreolus capreolus)[END_REF][START_REF] Van Wieren | Digestive strategies of ruminants and nonruminants[END_REF]; concentrates: [START_REF] Holand | Seasonal dynamics of digestion in relation to diet quality and intake in European roe deer (Capreolus capreolus)[END_REF], this study retained shorter than large, Ce-or La-labelled fibres [START_REF] Schwarm | Excretion patterns of fluid and different sized particle passage markers in banteng (Bos javanicus) and pygmy hippopotamus (Hexaprotodon liberiensis): two functionally different foregut fermenters[END_REF][START_REF] Lechner | Differential passage of fluids and different-sized particles in fistulated oxen (Bos primigenius f. taurus), muskoxen (Ovibos moschatus), reindeer (Rangifer tarandus) and moose (Alces alces): rumen particle size discrimination[END_REF]) can be regarded as very reliable because they demonstrate a difference opposite to the one expected based on the type of mordant alone.

Effect of dry matter intake on mean retention time-comparison with literature data Animals ingested on average 48±5 g kg -0.75 d -1 , which is in agreement with the intake of a similar pelleted food in the study of [START_REF] Holand | Seasonal dynamics of digestion in relation to diet quality and intake in European roe deer (Capreolus capreolus)[END_REF] (43±12 g kg -0.75 d -1 , n=3; Table 3). However, the MRT of Ce-labelled fibre in roe deer from our study was significantly longer (1.8-fold) than in the study of [START_REF] Holand | Seasonal dynamics of digestion in relation to diet quality and intake in European roe deer (Capreolus capreolus)[END_REF] (37±6 h vs. 21±2 h, Student´s t-test, p= 0.008, N=4 each), although relative DMI did not differ (Student's t test, p=0.493, N=4 each; Table 3). Usually, MRT decreases with increasing relative DMI, as seen in roe deer fed a roughage-only diet (Fig. 2, data from Holand 1994;[START_REF] Van Wieren | Digestive strategies of ruminants and nonruminants[END_REF]. However, this is not the case on a pelleted concentrate diet (for statistics, see Fig. 2 legend). Possible explanations for the shorter MRT in the study of Holand 1994 compared to our study are the chemical composition of the diet (ADF, 5% vs. >7%), the environmental conditions (4-vs. 28-m 2 enclosure size) and the type of fibre labelled (oat grain vs. grass hay).

In the study of [START_REF] Behrend | Seasonal faecal excretion, gut fill, liquid and particle marker retention in mouflon Ovis ammon musimon, and a comparison with roe deer Capreolus capreolus[END_REF], the same type of fibre was mordanted as in our study (grass hay), with resulting comparatively short MRT of 24 h (Table 3). DMI was not assessed by [START_REF] Behrend | Seasonal faecal excretion, gut fill, liquid and particle marker retention in mouflon Ovis ammon musimon, and a comparison with roe deer Capreolus capreolus[END_REF]; however, the faster excretion of Cr-mordanted particle markers in their study may be attributed to the considerable amount of available roughage (natural vegetation of the 600-m 2 enclosure supplemented with fresh and dried browse) with correspondingly accelerated digesta passage (cf. Fig. 2).

To conclude, Ce and La showed the same excretion pattern and can therefore be recommended as mordants in studies assessing the passage kinetics of different particle sizes.

Fig. 1

 1 Fig.1Excretion pattern for 2-mm hay particles (labelled either with cerium (Ce), lanthanum (La) or chromium (Cr)) in roe deer (animal 4). Marker concentrations adjusted for visual comparability. Note: Ce and La marker were ingested simultaneously as a pulse dose within 3 h. Cr marker uptake was 25 h prior to Ce and La; it was ingested within 1 h. The animal had a dry matter intake during the 6 days of the trial of 46± 2 g kg -0.75 day -1 . Intake the day prior to Cr marker feeding was 42 g kg -0.75 day -1 Table 2 DMI (mean ± SD) and MRT of 2-mm hay particles in the gastrointestinal tract of four roe deer (C. capreolus) studied in June 2006 at the field research station of the Leibniz Institute for Zoo and Wildlife Research Animal Age (years) Sex Body mass (kg) DMI of the respective food

  were labelled either with Ce, La or Cr. Ce and La markers were ingested simultaneously. Cr marker was ingested 21 h (animals 1-2) and 25 h (animals 3-4) prior to Ce and La a Individuals were offered on average 440 g DMday -1 pelleted compound feed for dairy calves and 68 g DMday -1 pelleted hay Table3MRT of feed particles in the gastrointestinal tract and relative DMI of roe deer (C. capreolus) from the literature and details of the animals and the methods used

Table 1

 1 MRT of similar-sized and simultaneously fed feed particles-either labelled with Cr or the lanthanide Ce, Dy and Yb-in the gastrointestinal tract of cattle and rabbit from the literature Different lanthanide-labelled fibres had the same MRT, whereas Cr-mordanted fibre retention time exceeds that of lanthanide-labelled fibres Source: 1 Huhtanen and Kukkonen (1995) 2 Ahvenjärvi et al. (2004), 3 Pond et al. (1989), 4 Mader et al. (1984), 5 Murphy et al. (1989), 6Beauchemin and Buchanan-Smith (1989), 7[START_REF] Goetsch | Ruthenium phenantrholine, dysprosium and ytterbium as particulate markers in beef steers fed an all-alfalfa hay diet[END_REF] Galyean (1983), 8 Gidenne and[START_REF] Gidenne | Rate of passage in the rabbit digestive tract: influence of marker dosing time, ileal cannulation and marker type[END_REF]

	Species	Particle type	Length of the particles mordanted (mm)	Metal salt used as mordant	MRT (h)		Source
					Ce	Dy	Yb	Cr
	Cattle	Grass hay	20-50	Na 2 Cr 2 O 7 , Yb-acetate			75	82	1
	Cattle	Grass silage	-(coarsely chopped)	Na 2 Cr 2 O 7 , Yb-acetate			39	46	2
	Cattle	Grass hay	-(masticated)	Na 2 Cr 2 O 7 , 169 Yb(NO 3 ) 3			59	61	3
	Cattle	Grass hay	60-120	Na 2 Cr 2 O 7 , YbCl 3			54	72	4
	Cattle	Rice straw	1	Na 2 Cr 2 O 7 , Yb-acetate			30	50	5
	Cattle	Alfalfa silage	-(large-chopped)	Na 2 Cr 2 O 7 , Yb-acetate			48	57	6
	Cattle	Alfalfa hay	25	DyCl 3 , YbCl 3		42	42	7
	Rabbit	Concentrates	0.05	CeCl 3 , YbCl 3	27		28	8

Table 2

 2 DMI (mean ± SD) and MRT of 2-mm hay particles in the gastrointestinal tract of four roe deer (C. capreolus) studied in June 2006 at the field research station of the Leibniz Institute for

		Body mass (kg) DMI of the respective food
	Zoo and Wildlife Research	Animal Age (years) Sex

  . MRT of different markers were compared with paired Student's t test and with general linear model repeated measurements (GLM RM). Comparison with literature data was performed by the use of an unpaired Student's t test. Monotonous associations between pairs of variables were measured by calculating Spearman's correlation coefficient. The significance level was generally set to α=0.05. Statistical calculations were performed with the SPSS 15 software (SPSS, Chicago, IL, USA).

Eur J Wildl Res (2011) 57:437-442

Acknowledgements This study was supported by grants from the Freie Universität Berlin (NaFöG) to AS and the German Science Foundation (DFG) to SO (OR 86/1-1). We thank A. Brandenburger, H. Barleben, J. Mahr, B. Baumann and U. Baum for their engaged support.


Céline Cosseau

Christoph Grunau
email: christoph.grunau@univ-perp.fr

6. Native Chromatin Immunoprecipitation (nChIP)

Native chromatin immunoprecipitation refers to a method that allows for identification and quantification of DNA that is associated with specific chromatin proteins without altering the structure of these proteins. The method has been used with great success in the past and has some advantages over the more widely used cross-linking chromatin immunoprecipitation. We describe here a protocol that was specifically optimized for low cell numbers.

Key Words

native chromatin; histones; immunoprecipitation 5. 15 U/!l Micrococcal nuclease (MNase) (EC 3.1.31.1) (USB 70196Y) in sterile 50% glycerol, aliquot to ~10!l and store at -20°C. Do not refreeze. MNase digests DNA between nucleosomes (4, 5).

6. 2% NaN 3 in water (store at 4°C) as preservative. Sodium azide is very toxic.

2.2.

Antibodies and Sepharose-Protein A

Introduction

In contrast to cross-linking chromatin immunoprecipitation (xChIP), native ChIP relies on relatively strong interaction between the target proteins and DNA under physiological salt conditions (1, 2). Histones are a typical example of such proteins, but nChIP has also successfully been used with transcription factors (3). In this protocol we focus on histone isoforms that are typical examples for a chromatin marking system. When histone-style proteins are targeted, we see three advantages of nChIP compared to xChIP: (i) the proteins remain in their native form and there is no danger that crosslinking fixes interactions that do not occur systematically in the cell; (ii) nChIP is 10-100 times more sensitive than xChIP and less starting material is required; and (iii) since enzymatic fragmentation of chromatin is used, no expensive equipment such as a sonicator is necessary.

Nevertheless, xChIP and nChIP are complementary, and ideally both approaches should be used in parallel.

Material

Enzymes and enzyme inhibitors

1. 100 mM Dithiothreitol (DTT) in distilled water, aliquot to 1 ml (store at -20°C). DTT is a reducing agent that prevents the formation of disulfide bonds in and between proteins.

2. 25 mM Phenylmethanesulfonylfluoride (PMSF) in isopropanol, 10 ml (store at -20°C). PMSF is a serine protease inhibitor.

3. Roche Complete Protease Inhibitor tablets (ref: 11 697 498 001) (store at 4°C) 4. 2.5 M Sodium butyrate in distilled water (Sigma B5887 1g) (store at 4°C). Sodium butyrate is an histone deacetylase (HDAC) inhibitor. Sodium butyrate is not very stable and should not be stored for more than 4 weeks in solution. The product is irritant and the solution has a nauseating odor, wear gloves! 1. aliquot antibodies on arrival to 2-4 !l and store at -20°C 2. preparation of sepharose-protein A:

a. weight 250 mg sepharose-protein A in 15 ml falcon tube b. wash with 10 ml sterile water c. centrifuge 10 min at 4000 rpm d. remove supernatant e. repeat washing step four times f. add sterile water to 5 ml g. add NaN 3 to 0.02% and store at 4°C 250 mg Protein A -sepharose swell to approximately 1 ml gel and bind about 20 mg human IgG.

You will need 50 !l of the homogeneously mixed sepharose-protein A per ChIP. Protein A has strong affinity to human, mouse and rabbit IgG, for antibodies raised in goat and sheep, sepharoseprotein G or protein A/G mixtures should be used. Paramagnetic sepharose particles are available but in our hands, background was on average 20 times higher than in a centrifugation-based separation.

Other stock solutions

1. 1 M KCl, autoclave 2. 5 M NaCl, filter to prevent formation of crystals later and autoclave f. before use, wash tube inside and outside with distilled water 3. centrifuges, stirrers, waterbath or similar, PCR machine, gel electrophoresis

Methods

Step 1: Testing the antibodies

By their very nature, immunoprecipitation methods rely on antibody-antigen interactions.

Specificity and the strength of this interaction will determine the quality of the ChIP experiment in terms of specificity and sensitivity. Since histones, the principle targets of nChIP, are highly conserved proteins, most laboratories will prefer to buy antibodies instead of producing their own.

Several monoclonal and polyclonal antibodies against histone isoforms are now commercially available. Some of them are "ChIP-certified", that is to say the manufacturer guaranties success of the immunoprecipitation if the "certified" animal or plant species are used. Nevertheless, antibody specificity and efficiency must always be tested. A simple Western blot gives a good indication of specificity. Already a straightforward protein extraction and separation on a standard SDS-PAGE will provide sufficient information (see Note 1). If bands can be detected outside the expected size range of the target proteins, and if the secondary antibody is not the origin of the problem, then antibodies from another source should be tested. Some companies provide antibody samples. While it cannot be excluded that antibodies that show unspecific binding in the Western blot are specific in the IP conditions, such antibodies should be avoided. The non-specific activity is probably due to immunogenic protein carriers used during immunization such as keyhole limpet hemocyanin KLH (6). An example is shown in figure 1. Ideally, a single band of the expected size should be visible.

In the example in figure 1, antibodies from supplier AB1 should not be used for ChIP since additional bands are detected. Antibodies from supplier AB2 are suitable. However, also absence of any detection does not indicate that the antibody does not work in immunoprecipitation. If the preimmune serum is available, IgG from this serum must be used as control in all steps of the here described procedure.

Step 2: Chromatin preparation

The principle of the procedure is shown in figure 2. The procedure follows tightly earlier protocols (7, 8) but has improved sensitivity and lower background (we use it routinely with less than 1500 Schistosoma mansoni larvae, i.e. roughly 150000 cells (9)). The following procedure describes nChIP for 3 samples and one control. One should use 10 6 -10 7 (at least 10 5 ) cells for each sample.

Cells can be aliquoted and stored at -80°C for up to 6 month or in liquid nitrogen for up to 12 month. However, it is preferable to use fresh biological material.

1. prepare a centrifuge for 10 or 50 ml tubes, up to 8500 rpm and cool it down to 4°C 2. preheat a water bath to exactly 37°C

3. prepare the solutions in tables 1-6 (always freshly)

Cell lysis and purification of nuclei

1. for culture cells or cell suspensions:

a. multiply 10 6 -10 7 cells with the number of antibodies to be used in immunoprecipitation (IP), for the control use the same amount of cells as for one antibody b. for each IP and for the control, transfer the corresponding amount of cells into a 2 ml Eppendorf tube and wash with 1 ml 150 mM NaCl (centrifuge 2500 rpm 10 min 4°C, remove supernatant)

2. for tissue samples: cut into small pieces and wash as above, use 10 6 -10 7 cells per antibody 3. resuspend pellets completely in 1 ml buffer 1 4. add 1 ml buffer 2 and homogenize for 3 min with Dounce (pestle A) on ice 5. put on ice 7 min 6. fill 8 ml buffer 3 into a 14 or 50 ml corex centrifugation tube, label the tubes appropriately 7. use 2 corex tubes for each sample 8. overlay the 8 ml buffer 3 with 1 ml cell suspension so that the tubes are ready for centrifugation 10 min after buffer 2 has been added to the cells, disturb the interface between buffer 3 and the sample a little bit with the pipette tip 9. if you don't use a swing-out rotor, mark the tubes on the exterior side (to know where to look for the nuclei) [START_REF] Maniatis | Molecular cloning : a laboratory manual[END_REF]. centrifuge 8500 rpm, 20 min, 4°C (ideally in a swing-out rotor)

11. remove the supernatant completely and carefully

Chromatin fragmentation with MNase

1. resuspend the two nuclei-containing pellets for each sample in 1 ml MNase digestion buffer 2. aliquot 500 !l of this suspension into two 1.5 ml Eppendorf tubes resuspend the pellet P1 in 100 !l dialysis buffer and dialyze overnight at 4°C against 50 ml dialysis buffer with gentle stirring. This dialysis step is important for liberation of chromatin fragments from the nuclear debris. For some cell types this step can be skipped, if no DNA can be detected in the pellet. In general, the dialysis step is however necessary (figures 3 and 4).

11. the next day, prepare a 2% 0.5x TBE agarose gel with 20 !l slots 12. thaw yesterdays supernatant S1, transfer dialyzed sample to Eppendorf tubes and...

Incubation with the antibody

When the antibodies have passed the initial Western blot test, the right antibody-to-chromatin ratio must be determined. For a given amount of chromatin, the antibody must be in excess, and the amount of immunoprecipitated DNA must not depend on the amount of antibody used. This titration procedure can be done with the below outlined procedure, using a constant quantity of chromatin and increasing amounts of antibodies. From a certain quantity of antibody on, the amount of immunoprecipitated DNA should remain constant (figure 5). For further experiments, an antibody concentration above this threshold must be used. If no threshold can be reached, the antibody is not suitable for IP since the amount of precipitated DNA will be a function of the used antibody amount. The advantage is that even if antibody concentrations are unknown, the suitable amount (in !l) can easily be determined. The procedure is costly since relatively large amounts of antibodies are consumed and much biological material is required, but it assures that in the following steps reproducible results can be achieved. Failure to determine the correct antibody to chromatin ratio would lead to many difficulties in subsequent experiments. transfer supernatant to a 1.5 ml tube. This is the bound fraction B.

To make sure, that the correct protein was immunoprecipitated, it is advisable to do a Western blot for fraction B. This is often not trivial since Protein A will leach into the SDS gel, and bands corresponding to the heavy and light chains of the antibody will be detected. Using beads coated with recombinant Protein G can in some cases provide a solution.

Step 5: Quantification of precipitated DNA

The amount of genomic region of interest (RoI) must be determined in the unbound and bound fractions of the control without antibody, and in the bound fraction with antibody (B). The unbound control fraction corresponds to the input (I), and the bound control fraction is the background (BG), i.e. DNA that sticks to the beads (figure 2). If no DNA can be detected in fraction B, the antibody unbound fraction UB must be analyzed, in order to exclude that DNA was entirely degraded.

Essentially, there are two possibilities to determine the quantity of RoI, either by comparing to an internal standard (reference locus method), or by determining the proportion of immunoprecipitated DNA compared to the input (% input recovery method).

Relative quantification (reference locus method)

In this case, in fraction B, I and BG a reference RoI and target RoI are quantified by quantitative PCR. The relative enrichment is calculated according to the following simplified formula:

enrichment factor = 2 -( ! Ct)
where !Ct = (Ct target -Ct reference ) B -(Ct target -Ct reference ) I

In this case it is assumed that the amplification efficiency of the PCR is close to 2 and identical for the two qPCR (for a detailed description of the underlying assumptions of the method and possible pitfalls see (11)). It could also be envisaged to use an efficiency-corrected comparative quantification or multiple reference genes.

By using a standard curve, the amount for each locus can be determined based on a dilution series of DNA of known concentration. The PCR efficiencies can be different. In this case: The standard curve method should be preferred if sufficient qPCR reactions can be performed simultaneously. The advantage of this quantification method is that target to reference DNA ratio in the same tube is measured. Even if DNA is lost during the purification process, the relative enrichment remains the same. Despite the relatively complex procedure, enrichment factors are very reproducible; in our hands standard errors are around 10%. The reference locus can for instance be in the body of a housekeeping gene. The enrichment factor described how much more chromatin modifications are associated with the target locus compared to the reference. A principal caveat of this quantification method is that it assumes that chromatin structure in the reference locus does not change. This is probably true for regions in the body of housekeeping genes, but it cannot be excluded, that under particular conditions, these regions change their chromatin status in parallel with the target locus. In this case, the relative quantification method would not be appropriate.

Absolute quantification (% input recovery method)

A solution could be to quantify directly the amount of precipitated target RoI (bound) and to compare it to the amount of target RoI in the input I (unbound fraction of control). In general, this ratio is expressed in % input recovery (12):

% input recovery = 100 * PCR-efficiency (Ct-input -Ct-bound)

The problem with this method is that DNA in two different tubes is compared (B and UB-C). If more DNA is lost during the preparation process in one tube, this will induce errors that cannot be detected. The method is sensitive to pipetting errors, requires careful standardization and, naturally, several technical and biological duplicates must be performed. Even then, day-to-day variations can be high (figure 6). Input recovery of BG should be " 0.1 %.

In conclusion, for a successful nChIP, it is indispensable to test the specificity of the antibodies and to determine the optimal amount by titration, prior to the experiment. For titration, a suitable target region in the genome must be known. For histone isoforms this will be relatively straightforward, for other chromatin proteins with restricted target regions, this might be more difficult.

Nevertheless, and for obvious reason, this positive control region is necessary. In the experiment itself, input recovery in the background must be low ("0.1%). In our hands, quantification via the reference locus method gives better reproducibility. The % input recovery method has relatively high day-to-day variations and, apart from the comparison to the background, it should only be applied if a reference locus cannot be used. mononucleosomes are preferred, however, these mononucleosomes should not be produced by prolonged digestion with MnaseI since this will introduce a bias in heterochromatin to euchromatin ratio. Other methods such as sucrose gradient centrifugation should be used (7).

In our hands, typical incubation times for 10 SDS-PAGE and incubated with antibodies against histone H3 (left) and histone H3 trimethylated at lysine 9 (middle). In each lane, the same amount of protein was applied. Antibodies from two different suppliers (AB1 and AB2) were used and revealed with a peroxidase coupled secondary antibody. On the right, only the secondary antibody was used as control. P2 is discarded because it contains cell debris that would interfere with the subsequent centrifugation steps. S2 fraction of 8 min digest was barely visible and the photo was software improved. The % input recovery corresponds to the proportion of DNA in a specific locus that is precipitated (fraction B), compared to the unbound fraction in the tube without antibody (input I). Differences in these fractions do not only reflect differences in binding vs. non-binding to the beads but can be introduced through experimental errors (e.g. pipetting errors, loss of precipitated material). In half of the shown experiments, input recovery in this locus was around 20%, but in other experiments it was higher. In one case it exceeded 80%, probably due to a loss of material during the purification of the input DNA that served as reference.

3. add 1

 1 !l MNase (15 U) and incubate 2-6 min at 37°C (see Note 2) 4. to stop the reaction add 20 !l 0.5 M EDTA to each 500 !l MNase digest and put the tube on ice 5. centrifuge 13000 g, 10 min, 4°C 6. transfer the supernatant to a new tube (S1) and keep the pellet (P1) on ice 7. quantify chromatin in S1 by measuring OD at 260 nm against MNase buffer (In general we find about 50 !g/ml DNA in the undiluted S1, OD260/280 values can be bad because there is a lot of protein in the solution. DNA quantification is therefore not precise but sufficient for reproducibility.) 8. store S1 at -20°C 9. humidify Slide-a-Lyzer with 50!l dialysis buffer 10.

  to new tubes and repeat the centrifugation and transfer steps 2 times, discard the pellets (P2). These triple centrifugations are important! They reduce the unspecific background! If you still observe a pellet after the third centrifugation, repeat the centrifugation step. 15. The final supernatants are fractions S1 (non-dialyzed) and S2 (dialyzed). 16. use 50 !l of S1 and S2 for phenol/chlorofrom extraction, centrifuge and load 20 !l of supernatant on a 2% 0.5x TBE agarose gel (100V, 25 min) to verify the presence of mono-to pentanucleosomes.

1 .

 1 use a standard phenol/chloroform extraction of DNA (10) in fractions B and UB 2. add 1 !l of a 20 g/l glycogen stock solution 3. add NaCl to 250 mM (26 !l and 52 !l for B and UB, respectively) and add 1 volume isopropanol 4. put overnight at -20°C or 30 min at -80°C 5. precipitate by centrifugation and wash with 70% ethanol 6. dry the pellet and resuspend in 40 !l 10 mM Tris/Cl 7. for single-copy loci use 2.5 !l of this DNA for PCR in 10 !l reactions (quantitative real-time PCR) or continue with massive sequencing (see chapter 22). In qPCR, single locus DNA should deliver Ct values of around 25 when starting with 150000 cells.

  enrichment factor = [ng target(B)/ng reference(B)] / [ng target(I)/ng reference(I)]

4 . Notes 1 . 2 . 1 - 5

 41215 Protein extraction and Western Blot: For high-resolution analysis of histones and histone isoforms, classical acid extraction and Triton-Acetic Acid-Urea (TAU) gels should be used[START_REF] Ryan | Separation of histone variants and posttranslationally modified isoforms by triton/acetic acid/urea polyacrylamide gel electrophoresis[END_REF]. For antibody testing we recommend the faster following method: Roughly 3x10 5 cells are suspended in denaturation buffer (0.2 % bromophenol, 10% sucrose, 3% SDS, and 0.2 M DTT, 62.5 mM Tris/Cl, pH 6.8) treated by sonication (e.g. 60% intensity of Vibra cell sonicator 75185, 6 times 15 sec, with cooling intervals on ice) and boiled 10 min at 95°C. Proteins are separated by conventional 15% SDS-PAGE and transferred for 1 hour to nitrocellulose membranes (e.g. Amersham RPN203D) by the semi-dry method. The membranes are blocked overnight at 4°C in blocking buffer (150 mM NaCl, 0.05% v/v Tween 20, 5% w/v fat-free dry milk, 20 mM Tris/Cl, pH 7.5) and incubated with the desired antibody for 2 hours in blocking buffer. The membranes are washed with washing buffer (150 mM NaCl, 0.05% v/v Tween 20, 20 mM Tris/Cl, pH 7.5) and incubated with a peroxidase-coupled secondary antibody for 30 min in blocking buffer. Bands are revealed by chemical luminescence (e.g. ECL Pierce) and direct exposure to x-ray film (Amersham EmNo.27304). For stripping, incubate the membrane at 50°C for 1 hour in stripping solution (2% SDS, 0.8% beta-mercaptoethanol, 62 mM Tris/Cl pH 6.8) in a tightly closed 50 ml tube and wash with distilled water several times until the smell of beta-mercaptoethanol has completely disappeared. A membrane can be used 3-4 times. Optimization of MNase digestion: The digestion time must be determined experimentally and can be different for each cell type and each MNase preparation. To determine the optimal digestion time, transfer every minute 100 !l of the reaction mix into Eppendorf tubes containing 10 !l 0.5 M EDTA on ice. Mix 50 !l of each fraction with 50 !l phenolchloroform, vortex, centrifuge and apply 20 !l of the upper phase on the 2% 0.5x TBE agarose gel. Separate the fragments (100V, 25-35 min) and observe after ethidiumbromide or other DNA staining. Optimal digestion is achieved when DNA fragments corresponding to nucleosomes are visible. For ChIP followed by massive sequencing (ChIP-Seq),

Figure 2 :

 2 Figure 2: Schematic representation of the nChIP procedure.

Figure 3 :

 3 Figure 3: PCR products separated on agarose-gels used for the detection of 28S rDNA genes in

Figure 4 :

 4 Figure 4: (A) Optimization of the MnaseI digestion process. DNA was extracted from the S1

Figure 5 :

 5 Figure 5: Example for the results of a titration experiment. In this case, antibody saturation was

Figure 6 :

 6 Figure 6: Frequency distribution of % input recovery for anti-acetyl H3K9 in the S.mansoni alpha-

  1. Prepare a dilution series of your chromatin in MNase buffer starting with 20 -40 !g chromatin DNA for histone ChIP and use 2-20 !g antibody (if the concentration is not known use 2 -20 !l antibody).

	9. mix for 10 min on a rotating wheel at 4°C
	10.	centrifuge 10 min 4000 rpm 4°C and pour off supernatant
	11.	add 10 ml washing buffer B, mix for 10 min on a rotating wheel at 4°C and
	2. Add appropriate amounts of stock solutions to generate the antibody incubation buffer (150 centrifuge 10 min 4000 rpm 4°C
	mM NaCl, 20 mM sodium butyrate, 5 mM EDTA, 0.2 mM PMSF, 20 mM Tris/Cl pH 7.5) by
	12.	pour off supernatant
	taking into consideration the amount of S1 and S2 and their respective buffers.
	13.	add 10 ml washing buffer C, mix for 10 min on a rotating wheel at 4°C and
	3. Dilute S1 and S2 in 1ml final volume of antibody incubation buffer.
	centrifuge 10 min 4000 rpm 4°C
	4. Add the appropriate amount of antibody (typically 4-8 !g). Add an equal volume to the
	14.	pour off supernatant
	control tube without antibody.
	15.	centrifuge 10 min 4000 rpm 4°C
	5. Incubate overnight at 4°C on a slowly rotating wheel.
	16.	remove remaining supernatant completely (centrifuge if necessary)
	17.	resuspend pellet in 500 !l elution buffer (table 8) at room temperature
		3.3.	Immunoprecipitation and DNA extraction
	18.	transfer to a 1.5 ml tube and incubate 15 min at room temperature on a rotating
	wheel		3.3.1.	Immunoprecipitation
	1. prepare 50 !l of sepharose-protein A for each tube 19. centrifuge 10 min, 11600 g, 18-20°C
	2. wash the beads to remove NaN 3 : short spin, remove supernatant and replace with equal 20.
	volume of sterile water
	3. add 50 !l of sepharose-protein A to each tube, including the control tube
	4. incubate at least 4 h at 4°C on a rotating wheel
	5. prepare washing buffers (10 ml / tube) and cool down to 4°C (table 7)
	6. centrifuge chromatin-antibody-beads mixture 10 min 4°C 11600 g
	7. keep the supernatant in a 2 ml tube. This is the unbound fraction UB.
	8. resuspend the pellet in approximately 1 ml washing buffer A and transfer into a 15 ml Falcon
	tube containing 9 ml washing buffer A

table 1 : 2x base buffer 1x

 1 6 cells and 37°C are 2-6 minutes. An example is shown in figure4A. In this case, liver tissue of a Syrian hamster (Mesocricetus auratus) was used. After 8 min, virtually all chromatin is digested to mononucleosomes. Figure4Billustrates the distribution of DNA fragments in the different fractions of the chromatin preparation. Fraction S2 is enriched in larger fragments. It should be noted that MnaseI incubation time influences also on the relative amount of DNA in the different fractions. This is shown in table 9. In this example, after 4 min MnaseI treatment, the distribution of different loci in the hamster genome is relatively homogenous. In contrast, after incubation for 8 min with MnaseI, distribution is uneven. Since the majority of the chromatin in S1 and : 60 mM KCl, 15 mM NaCl, 5 mM MgCl 2 , 0.1 mM EDTA, 15 mM Tris/Cl, pH 7.5

	6 ml	1 M KCl
	1.5 ml	1 M Tris/Cl
	0.3 ml	5 M NaCl
	0.5 ml	1 M MgCl 2
	20 µl	500 mM EDTA
	to 50 ml with autoclaved distilled water, put on ice
	2 Roche protease inhibitor tablets
	table 2: buffer 1
	0.3 M sucrose, 5 mM CH 3 CH 2 CH 2 COONa, 0.1 mM PMSF, 0.5 mM DTT, 60 mM KCl, 15
	mM NaCl, 5 mM MgCl 2 , 0.1 mM EDTA, 15 mM Tris/Cl, pH 7.5
	2.58 g	sucrose
	12.5 ml	2x base buffer
	50 µl	2.5 M sodium butyrate
	100 µl	25 mM PMSF
	125 µl	100 mM DTT
	to 25 ml with autoclaved distilled water, put on ice
	table 3: buffer 2
	0.3 M sucrose, 5 mM CH 3 CH 2 CH 2 COONa, 0.1 mM PMSF, 0.5 mM DTT, 0.8% (v/v)
	NP40, 60 mM KCl, 15 mM NaCl, 5 mM MgCl 2 , 0.1 mM EDTA, 15 mM Tris/Cl, pH 7.5
	10 ml	buffer 1
	put on 37°C to allow Nonidet P-40 (NP40) to be pipetted into the buffer
	80 µl	NP40 or replacement product (Sigma-Aldrich has replaced
		Nonidet P-40 with a nonionic, non-denaturing substitute detergent
		(Fluka 74385). The principle is that the detergent will destroy the
		cytoplasmic membrane but not the nuclear membrane.

table 3 : buffer 2

 3 Centrifugation separates the intact nuclei by precipitation.) put on 37°C to fully dissolve NP40, mix well but do not vortex and put on ice

table 4 : buffer 3 for 3 samples and 1 control

 4

	1.2 M sucrose, 5 mM CH 3 CH 2 CH 2 COONa, 0.1 mM PMSF, 0.5 mM DTT, 60 mM KCl, 15
	mM NaCl, 5 mM MgCl 2 , 0.1 mM EDTA, 15 mM Tris/Cl, pH 7.5
	20.55 g	sucrose
	25 ml	2x base buffer
	100 µl	2.5 M sodium butyrate
	200 µl	25 mM PMSF
	250 µl	100 mM DTT
	to 50 ml with autoclaved distilled water, put on ice
	table 5: MNase digestion buffers
	0.3 M sucrose, 5 mM CH 3 CH 2 CH 2 COONa, 0.2 mM PMSF, 4 mM MgCl 2 , 1 mM CaCl 2 , 50
	mM Tris/Cl, pH 7.5
	1.1 g	sucrose
	0.5 ml	1M Tris/Cl
	80 µl	25 mM PMSF
	40 µl	1 M MgCl 2
	20 µl	2.5 M sodium butyrate
	10 µl	1 M CaCl 2 (essential for the enzyme)
	to 10 ml with autoclaved distilled water, put at 37°C

table 6 :

 6 Dialysis buffers 5 mM CH 3 CH 2 CH 2 COONa, 0.2 mM PMSF, 0.2 mM EDTA, 1 mM Tris/Cl, pH 7.5

	10-20 ml	autoclaved distilled water
	400 µl	25 mM PMSF
	100 µl	2.5 M sodium butyrate
	50 µl	1M Tris/Cl
	20 µl	500 mM EDTA
	to 50 ml with autoclaved distilled water, put on ice

table 7 : Washing buffers A,B and C (example 100 ml)

 7

	5 mM CH 3 CH 2 CH 2 COONa, 10 mM EDTA, 75 mM (A) / 125 mM (B) / 175 mM (C) NaCl,
	50 mM Tris/Cl, pH 7.5
	1.5 ml (A)	5 M NaCl
	or 2.5 ml (B)
	or 3.5 ml (C)
	200 µl	2.5 M sodium butyrate
	5 ml	1M Tris/Cl
	2 ml	500 mM EDTA
	to 100 ml with autoclaved distilled water, put on ice
	table 8: Elution buffer
	1% SDS, 50 mM NaCl, 5 mM EDTA, 20 mM sodium butyrate, 0.1 mM PMSF, 20 mM
	Tris/Cl pH 7.5
	500 µl	20% SDS
	200 µl	1M Tris/Cl
	100 µl	5 M NaCl
	80 µl	2.5 M sodium butyrate
	40 µl	25 mM PMSF

Table 9 : % DNA in the different fractions after 4 and 8 min Mnase I digest at 37°C

 9 Figure 1: Luminescence-revealed Western blots of S.mansoni protein extracts, separated on 15%

		4 min Mnase I	8 min Mnase I
	Locus	S1 S2 S1+S2 P2 S1 S2 S1+S2 P2	Quantification method
	total DNA	49 18	67	33 37 15	52	48	nanodrop
	P53 (Genbank U08134) 74 15	89	11 13 69	82	18	qPCR (176 bp)
	5S (Genbank J00063)	66 8	74	25 11 4	15	85	qPCR (101 bp)
	Heterochromatic
	repeat(Genbank	79 6	85	15 10 3	13	86	qPCR (151 bp)
	AB185090)

S2 is digested to mononucleosomes of around 150 bp, this might also reflect problems in PCR amplification for larger primer distances.

5.