title: Illumina HiSeq 2500 paired end sequencing; MNase-Seq of isolated mono-nucleosomes from strains W303 (wild-type) and htz1∆.
design: MNase-Seq of isolated mono-nucleosomes from strains W303 (wild-type) and htz1∆.
library_construction_protocol: Purification of yeast genomic chromatin Nuclei were prepared by an adaptation of the protocol described in (Almer and Horz 1986). Six litres of YPD were inoculated with 300 ml saturated starter culture in the morning to harvest cells grown to mid-log phase (4-6 cells/ml) in the early afternoon. Cells were pelleted at 3,500 x g for 10 minutes and resuspended in 240 ml (50 mM Tris pH 7.5, 40 mM DTT) for gentle shaking at 30˚C for 15 minutes. Cells were pelleted at 11,000 x g for 5 minutes (JA-10 rotor) and resuspended in 60 ml 0.5x YPD/1 M sorbitol at room temperature (from here on all buffers were supplemented with standard protease inhibitors and 5 mM sodium butyrate and 5 mM trichostatin A as deacetylase inhibitors). For spheroblasting, 100 mg of zymolase 20T were added, and the cells shaken slowly for 30-50 minutes at 30˚C, until the OD600 of a 1:100 dilution in 1% SDS was less than 5% of the initial OD600 (e.g. 0.034 vs. initial 0.330). It is important not to overdigest, since the histone tails are sensitive to proteolysis. Zymolysis was stopped by addition of 100 ml cold 1 M sorbitol, and all subsequent operations were performed in the cold. Spheroblasts were pelleted and rinsed twice by centrifuging at 1,500 x g for 5 minutes (JA-10 rotor) and washing twice with 200 ml cold 1 M sorbitol. The rinsed spheroblasts were resuspended in 100 ml Ficoll Buffer (18% Ficoll 400, 20 mM potassium phosphate pH 6.8, 1 mM MgCl2, 0.25 mM EDTA, 0.25 mM EGTA) to lyze cells and release the nuclei. Nuclei were centrifuged at 45,000 x g for 20 minutes (Ti-45 rotor), then resuspended in 30 ml SucB1 (0.34 M sucrose, 20 mM Tris pH 7.5, 50 mM KCl, 5 mM MgCl2). The resuspended nuclei were then layered on 15 ml SucB2 (1.7 M sucrose, 20 mM Tris pH 7.5, 50 mM KCl, 5 mM MgCl2) and centrifuged at 115,000 x g for 30 minutes (SW-32 rotor) to further clean up the nuclei. These were resuspended in 10 ml SucB1, aliquoted to ~12 x 1 ml, and centrifuged at 18,000 x g for 1 minute. Supernatants were discarded, and pelleted nuclei frozen in liquid nitrogen for storage. Frozen nuclei could be stored indefinitely at -80˚C.    RSC-dependent nucleosome disassembly assayThe assay was adapted from the protocol described in (Lorch et al. 2006), except that DNA was stained with ethidium rather than by radioactive labelling. Each reaction was performed in 20 µl volumes, with 6-10 µl nucleosomes, 800 ng RSC, 2 µg Nap1, 1 mM ATP, 20 mM potassium acetate pH 7.6, 15 mM Hepes pH 7.9, 3 mM MgCl2, 75 µg/ml bovine serum albumin, and protease inhibitors (Rsc:nucleosome molar ratio ~1:4 – 1:2). After incubation for 15 minutes (replicate 1) or  20 minutes (replicate 2) at 30˚C, 1 µl of ~2 mg/ml plasmid DNA was added to capture free RSC, followed by the addition of 5 µl 30% glycerol. At this point, the mixture could be flash-frozen in liquid nitrogen and stored indefinitely at -80˚C. For native gel-electrophoresis, the thawed mixture was loaded on a pre-cooled 2% agarose gel in 0.5x TBE, then run for ~30 minutes at 50 V and ~1.5 hours at 100 V. Bands were stained by soaking the gel in 1 µg/ml ethidium bromide for one hour, destained with water for 30 minutes and bands analyzed with UV light. In our experience, bands were generally very weak, with their intensity varying greatly depending on the concentration of nucleosomes. After analysis, gel slices were excised, and DNA extracted using a commercial kit (Life Technologies GeneJET), to be eluted into 50 µl elution buffer. This DNA was suitable for sequencing or qPCR analysis. Nucleosomes were prepared from nuclei over two days. On the morning of day 1, one aliquot of nuclei was thawed and resuspended in 500 µl cold MNase Buffer (50 mM NaCl, 13 mM Tris pH 8.0, 6.4 mM CaCl2, 0.2 mM EDTA, 0.2 mM EGTA), before adding 10 µl 0.1 M CaCl2 and protease and deacetylase inhibitors as above. Resuspended nuclei were pre-heated for 90 seconds at 37˚C, and digestion performed with ~40,000 U MNase (~20 µl of 2,000 U/µl from New England Biolabs) for 2 minutes at 37˚C. Digestion was stopped by addition of 25 µl 0.5 M EGTA and placing of the tube on ice for 2 minutes. Optimal MNase concentrations were determined in a trial digestion for each batch of nuclei, by testing five different concentrations of MNase with 100 µl resuspended nuclei and choosing the concentration that yields mostly mono-nucleosomes, but still results in formation of some oligo-nucleosomes. It is crucial that the digestion be performed as fast as possible, and that all other steps be performed on ice or in the cold room to avoid proteolysis of histone tails. After digestion, the incubated nuclei were spiked with more protease inhibitors, then centrifuged at 18,000 x g for 1 minute. The supernatant containing mono-nucleosomes was kept and centrifuged one more time to remove remaining nuclei. The solution should be brown to yellow in colour, and clear. For DEAE chromatography, we used a self-poured 1.2 cm x 7 cm column (6 ml bed volume) equilibrated in DEAE-NucB-20 (20 mM NaCl, 10 mM Tris pH 7.5, 1.5 mM MgCl2, 10% glycerol). All 500 µl of nuclear lysate were loaded at 0.3 ml/min, and elution was performed in a 20-800 mM NaCl gradient in DEAE-NucB at 0.2 ml/min over 40 minutes. Chromatin eluted at about 400 mM in the second half of the major peak. The high salt concentration of ~400 mM used for elution from DEAE ensured that DNA-binding transcription factors were released and washed off, while the gradient centrifugation served to remove contaminants and separate free nucleosomes from nucleosomes bound by other proteins. Before proceeding with the sucrose gradient, fractions containing chromatin were identified with a quick agarose gel analysis by extracting DNA from 20 µl using the Qiagen PCR purification kit, performing a brief digestion with RNAse A and separating bands on a 1.5% agarose gel. Fractions containing chromatin were pooled and loaded on a staggered 20-45% sucrose gradient consisting of 5 ml each of 45, 40, 35, 30, 25 and 20% sucrose in Sucrose Buffer (30 mM NaCl, 10 mM Tris pH 7.5, 1 mM EDTA, 1 mM EGTA). Ultracentrifugation was performed at 200,000 x g for 26 hours (SW41 rotor). Fractions of ~500 µl were then collected by piercing the bottom of the tube (~8 drops per fraction, ~20 fractions total). To identify the fractions containing DNA, 15 µl of each fraction was incubated with ~30 µg RNAse A for 20 minutes at 37˚C, before adding 3 µl loading buffer containing 1% SDS, and separating bands on a 1.5% agarose gel. Fractions containing mono-nucleosomes were pooled, distributed into 25 or 50 µl aliquots, flash-frozen in liquid nitrogen, and stored at -80˚C. Final recoveries were ~1 to 1.5 ml at concentrations of ~20 ng/µl, corresponding to 20-30 µg of DNA. Each aliquot was thawed only once. We recommend testing the integrity of the histone tails by performing a western blot for histone H3 and checking for the absence of a lower band corresponding to proteolyzed histone. DNA concentrations in the final material were determined by qPCR using a standard curve of two different PCR products of known concentration that covered a part of the genome (primers P764/765 and P788/789).
attributes.tag: Experimental Factor: treatment
attributes.value: none
attributes.tag: Experimental Factor: fraction
attributes.value: DNA