title: GSM7329078: W306; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: Yeast cultures of 50 mL in complete medium were collected at OD600 = 0.4–0.6 for each condition. The cells were crosslinked with 1% formaldehyde for 10 min and quenched by adding 125 mM glycine for 5 min at room temperature. Cells were then washed with ice-cold HBS (50 mM HEPES-Na pH:7.5, 140 mM NaCl) and resuspended in 0.6 ml of ChIP lysis buffer (50 mM HEPES-Na pH:7.5, 140 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% sodium deoxycholate) supplemented with 1 mM PMSF and 1× protease inhibitor cocktail (Roche; Cat# 04693132001). The cells were broken using Zirconia/Silica beads (BioSpec) and lysates were centrifuged at 13,000 rpm for 30 min at 4°C. Following centrifugation, the pellets were resuspended in 300 μl ChIP lysis buffer containing 1 mM PMSF and sonicated for 15 min (30s ON–60s OFF) in a Bioruptor (Diagenode). The lysates were centrifuged at 7000 rpm for 15 min at 4°C, following which primary antibodies were added to the supernatant and incubated for 1 h at 4°C. ChIP was performed using Abcam ab5131 for RNAPII. Magnetic beads coupled to IgG against rabbit (Dynabeads™ M-280 Sheep Anti-Rabbit; Thermofischer; Cat# 11203D) were washed three times with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) containing 0.5% BSA and added to the lysates (30 μl of beads/300 μl of cell lysate). The samples were incubated for 2 h at 4°C. The beads were washed twice with AT1 buffer (50 mM HEPES-Na pH: 7.5, 140 mM NaCl, 1 mM EDTA, 0.03% SDS), once with AT2 buffer (50 mM HEPES-Na pH: 7.5, 1 M NaCl, 1 mM EDTA), once with AT3 buffer (20 mM Tris–Cl pH: 7.5, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate) and twice with TE buffer (10 mM Tris, 1 mM EDTA). The chromatin was eluted from the beads by resuspension in TE containing 1% SDS and incubation at 65°C for 10 min. The eluate was transferred to new Eppendorf tubes and incubated overnight at 65°C to reverse the crosslinks. The DNAs were purified using the MinElute PCR Purification Kit (Qiagen; Cat# 28004). DNA libraries were prepared using TruSeq ChIP Sample Preparation Kit (Illumina) according to manufacturer's specifications. The libraries were sequenced using an Illumina HiSeq 2500 platform at the Institute of Genetics and Genomics of Geneva (iGE3; http://www.ige3.unige.ch/genomics-platform.php) and the reads were mapped to the sacCer3 genome assembly using HTSStation60 (read densities were calculated using shift: 100, extension: 50 bp). All densities were normalized to 10M reads. The signal was quantified for each gene between the transcription start site (TSS) and transcription termination site (TTS). All data from publicly available databases were mapped using HTS Station (http://htsstation.epfl.ch;). TruSeq ChIP library preparation kit (Illumina). Post amplification libraries were size selected at 250-450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit.