title: GSM8640732: PolII ChIP-seq of XVIfMmycot1 strain +thiolutin (input), rep1; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: Cells were fixed using 4mL of fixative solution (50 mM Tris-HCl, pH 8.0; 100 mM NaCl; 0.5 mM EGTA; 1 mM EDTA; 30% (v/v) formaldehyde) for 30 min at room temperature (RT) with rotation. The fixative was quenched with 2mL of 2.5M glycine (RT, 5 min with rotation). The cells were then harvested by centrifugation at 3,500 rpm for 3 min and washed with ice-cold PBS. The cells were then resuspended in 300 mL of ChIP lysis buffer (50 mM HEPES KOH, pH 8.0; 140 mM NaCl; 1 mM EDTA; 1% (v/v) Triton X-100; 0.1% (w/v) sodium deoxycholate; 1 mM PMSF; 2X Complete protease inhibitor cocktail (Roche)) and transferred in 2mL tubes containing glass beads (ozyme, P000913-LYSK0-A.0) before mechanical cells lysis. The soluble fraction was isolated by centrifugation at 2,000 rpm for 3min then transferred to sonication tubes (Covaris milliTUBE 1ml, 520135) and samples were sonicated to produce sheared chromatin with a size range of 200-1,000bp using a Covaris sonicator. After sonication the samples were centrifuged at 13,200 rpm at 4°C for 20min and the supernatant was transferred into 700µL of ChIP lysis buffer. 80µL (27µl of each sample) of the supernatant was removed (termed 'whole cell extract [WCE] sample') and store at -80°C. 5ug of antibody was added to the remaining supernatant which is then incubated overnight at 4°C (wheel cold room). 50µL of protein G Dynabeads or protein A Dynabeads was then added and incubated at 4°C for 2h. Beads were washed 2 times with ChIP lysis buffer, 3 times with high salt ChIP lysis buffer (50mMHEPES-KOH, pH 8.0; 500 mM NaCl; 1 mM EDTA; 1% (v/v) Triton X-100; 0.1% (w/v) sodium deoxycholate;1 mM PMSF), 2 times with ChIP wash buffer (10 mM Tris-HCl, pH 8.0; 0.25MLiCl; 0.5% NP-40; 0.5% sodium deoxycholate; 1mM EDTA;1 mMPMSF) and 1 time with TE pH7.5. The immunoprecipitated chromatin was then eluted by incubation in 120µL TES buffer (50 mMTris-HCl, pH 8.0; 10 mM EDTA; 1% SDS) for 15min at 65°C and the supernatant is collected termed 'IP sample'. The WCE samples were mixed with 40µL of TES3 buffer (50 mM Tris-HCl, pH 8.0; 10 mM EDTA; 3% SDS). ALL (IP and WCE) samples were de-cross-linked by incubation at 65°C overnight. RNA was degraded by incubation with 2µL RNase A (10 mg/mL) for 1h at 37°C. Proteins were removed by incubation with 10µL of proteinase K (18 mg/mL) for 2h at 65°C. DNA was purified by a phenol/Chloroform extraction. The triplicate IP samples were mixed in 1 tube and libraries for IP and WCE samples were prepared using Invitrogen TM Collibri TM PS DNA Library Prep Kit for Illumina and following manufacturer instructions. Paired-end sequencing on an Illumina NextSeq500 (2x35 bp) was performed.