GSM5113889: 190722L68_MS522_SWI4-V5-ChIP; Saccharomyces cerevisiae; ChIP-Seq
Cells were fixed with 1% formaldehyde (30 minutes), quenched with 0.125 M glycine (5 minutes), washed twice in cold PBS, pelleted, snap-frozen and stored at −80°C. Pellets were lysed in 300 µL FA lysis buffer (50 mM HEPES–KOH pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM PMSF, Roche protease inhibitor) with ∼1 mL ceramic beads using a Fastprep-24 (MP Biomedicals). The lysate was then collected and sonication to ~200bp fragments. Cell debris was then pelleted and the supernatant retained for ChIP. For each ChIP reaction, 30 µL Protein G Dynabeads (Invitrogen) were blocked (PBS + 0.5% BSA), prebound with 10 µL anti-V5 antibody (SV5-Pk1, BioRad Cat# MCA1360G) or 10 µL anti-FLAG antibody (M2, SIGMA Cat# F1804) and washed once with PBS before incubation with supernatant (4°C, overnight). Dynabeads were then washed (5 minutes per wash) twice in FA lysis buffer, twice in high-salt (500 mM NaCl) FA lysis buffer, twice in ChIP wash buffer (10 mM TrisHCl pH 7.5, 0.25 M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF) and once in TE wash buffer (10 mM TrisHCl pH 7.5, 1 mM EDTA, 50 mM NaCl). DNA was eluted in ChIP elution buffer (50 mM TrisHCl pH 7.5, 10 mM EDTA, 1% SDS) at 65°C for 15-20 minutes. Eluted DNA was incubated to reverse crosslinks (65°C, 5 hours), before treatment with RNAse A (37°C, 1 hour) and then Proteinase K (65°C, 2 hours). DNA was purified using the ChIP DNA Clean & Concentrator kit (Zymo Research). Indexed sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep kit (NEB, # E7645) and pooled before paired-end (2x150 bp) Illumina sequencing