GSM7910868: Scc3_Smc3PK_IN_N1; Saccharomyces cerevisiae; Nakaseomyces glabratus; ChIP-Seq
50 OD600 units of Saccharomyces cerevisiae cells were mixed at a 1:5 ratio with Candida glabrata cells in which corresponding cohesin subunits were fused to a Pk epitope tag[Hu, 2015 #1305], and broken in 700 ml Lysis Buffer (50mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% Sodium Deoxycholate) supplemented with protease inhibitors by glass bead rupture. The lysate was retrieved by centrifugation for 1 minute at 2000 rpm and the supernatant then removed after centrifugation for 10 minutes at maximum speed in a benchtop centrifuge. The pellet was resuspended in 1 ml lysis buffer and sonicated to reach an average DNA fragment size of 500 bp. Cohesin was precipitated using and a-Pk antibody (clone SV5-Pk1, Bio-Rad) and protein A-coupled Dynabeads for 2 hours. Beads were washed 3 times with Lysis Buffer, 3 times with Lysis Buffer supplemented with 500 mM NaCl and 2 times with Wash Buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate). The samples were resuspended in 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS including 3 mg/ml proteinase K and 2 mg/ml RNase A) and de-crosslinked overnight at 65 °C. DNA was purified by phenol-chloroform extraction and the aqueous fraction was further cleaned using DNA Clean & Concentrator-5 with Zymo-Spin Columns (Zymo Research). Libraries were built with NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) following the manufacturer instruction. Sequencing was performed on an Illumina NovaSeq 6000 (100 bp paired).