GSM3100408: H3_ChIP-seq_spt16-197_rep1; Saccharomyces cerevisiae; ChIP-Seq
Crosslinked cells were collected by centrifugation and washed twice with 1 X TBS (20 mM Tris-HCl pH 7.5, 150 mM NaCl). Cells were then resuspended in 700 uL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin). The same number of OD600 units was used for all samples. A fixed amount of crosslinked S. pombe cells (representing 10% of the S. cerevisiae cells) was added to the S. cerevisiae cells prior to cell lysis. This spike-in control was used to normalise samples. Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a micro probe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation. 20 uL of the chromatin sample were saved for control of the average chromatin fragment size. For H3K4me3 and H4 immunoprecipitations, 25% (150 uL) of the chromatin sample, completed to 600 uL with Lysis buffer, was used. For H3K56ac and H3 immunoprecipitations, 50% (300 uL) of the chromatin sample, completed to 600 uL with Lysis buffer, was used. 6 uL (1%) of the diluted chromatin samples were saved as Input sample. 12.5 uL of Protein G Dynabeads (Thermo Fisher Scientific, 10004D) pre-coupled with the indicated rabbit polyclonal antibody were added to the 600 uL of diluted chromatin sample and incubated overnight at 4°C. Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Immunoprecipitated chromatin was eluted and reverse-crosslinked with 50 uL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Input and fragment size control samples were also added with 50 uL TE/SDS and incubated overnight at 65°C to reverse crosslinking. Eluted chromatin as well as Input and fragment size control samples were treated with RNase A (345 uL TE, 3 uL RNAse A 10 mg/mL, 2 uL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 uL 10% SDS, 7.5 uL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 100 % EtOH. Precipitated DNA was resuspended in 50 uL of TE and subjected to a 1.7X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers' instructions. Samples were eluted with 40 uL of elution buffer (10 mM Tris pH 8.0).   The sample used for determining the average chromatin fragment size was analysed on an Agilent 2100 Bioanalyzer instrument using Agilent High Sensitivity DNA Kit.  DNA concentration of the ChIP and Input samples was determined by qPCR using a standard curve made with the fragment size control sample. 3-10 ng of ChIP and Input DNA were used for ChIP-seq library preparation as follow. The ends of DNA were repaired using T4 DNA polymerase (NEB, M0203L) and T4 polynucleotide kinase (NEB, M0201S) at 12˚C for 30 min. Repaired DNA was then subjected to a 1.7X cleanup using KAPA Pure Beads before dA tailing using Klenow Fragment (3'→5' exo-) (NEB, M0212M) at 37˚C for 30 min. After a 1.8X cleanup with KAPA Pure Beads, the A-tailed DNA was ligated to index adapters (Roche, SeqCap Adapter kit A, 12 adapters (07141530001) and SeqCap Adapter kit B, 12 adapters (07141548001)) using T4 DNA ligase (ThermoFisher, 15224041) at room temperature for 60 min. The ligated DNA was then subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.52X-1X) leading to fragments in the 200-600 bp range. Libraries were PCR amplified with 12-13 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and cleaned up using 1X KAPA Pure Beads. Libraries were qualified on Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA Kit and quantified by qPCR using NEBNext Library Quant Kit for Illumina. Equal molarity of each library was pooled (12 libraries per pool) and subjected to sequencing on an Illumina HiSeq 2500 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp single-end reads.
GEO Accession
GSM3100408