title: GSM3670753: L485-ChIP-Diamide-0min-V5_Input; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: Cells were fixed with 1% formaldehyde for 20 minutes (Rpb1-CTD and Rbp1-CTD-S2P ChIP) or 30 minutes (Hsf1-V5 ChIP) and quenched with 0.125 M glycine for 5 minutes. A total of ~50 ODs of cells were used per Rpb1-CTD or Rpb1-CTD-S2P ChIP and ~300 ODs per Hsf1-V5 ChIP. Fixed cell were washed 2X in cold 1X PBS, pelleted and stored at -80C. Pellets were lysed in 300 uL 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 on a Fastprep-24 (MP Biomedicals). The entire lysate was then collected and adjusted to 1 mL with FA lysis buffer before sonication with a 1/8' microtip on a Q500 sonicator (Qsonica) for 14 minutes (10 seconds on, 20 seconds off). The sample tube was held in a -20C 80% ethanol bath throughout sonication to prevent sample heating. After sonication, cell debris was pelleted and the supernatant was retained for ChIP. For each ChIP reaction, 30 uL Protein G Dynabeads (Invitrogen) were blocked (PBS + 0.5% BSA), prebound with 5-10 uL antibody (8wG16 Rpb1-CTD, Abcam cat # ab817); 3E10 Rpb1-CTD-S2P, Milipore cat \# 04-1571-1) or SV5-Pk1 (anti-V5, BioRad cat # MCA1360G)) and washed 1X with PBS before incubation with supernatant (4C, overnight). Dynabeads were then washed (5 minutes per wash) 3X in FA lysis buffer, 3X in high-salt FA lysis buffer (50 mM Hepes–KOH pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM PMSF), 1X in ChIP wash buffer (10 mM Tris–HCl pH 7.5, 0.25 M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF) and 1X in TE wash buffer (10 mM Tris–HCl pH 7.5, 1 mM EDTA, 50 mM NaCl). DNA was eluted from the beads in ChIP elution buffer (50 mM Tris–HCl pH 7.5; 10 mM EDTA; 1% SDS) at 65C for 20 min. Eluted DNA was incubated at 65C overnight to reverse crosslinks, before treatment with RNAse A (37C, 1 hour) and then Proteinase K (65C, 2 hours). DNA was purified using the ChIP DNA Clean & Concentrator kit (Zymo Research). Sequencing libraries were generated using the NEBNext Ultra II DNA Library Prep kit  (NEB Cat # E7645) and sequenced on a Illumina NextSeq instrument as 2x36mers or as 2x75mers.
attributes.tag: GEO Accession
attributes.value: GSM3670753