GSM6052388: scc2_IAA_tc_1; Saccharomyces cerevisiae; ChIP-Seq
Cells were fixed in 1% formaldehyde for 30 min at room temperature, then quenched with glycine (final concentration 0.125 M) for 5 min. Cells were washed twice with cold PBS, frozen in liquid nitrogen, and stored in −80°C until used. Cells were lysed by bead beating at 4°C in FA-Lysis SDS buffer (50 mM Hepes (pH7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.2% SDS, protease inhibitors). The lysate was sonicated using a Covaris SS20 sonicator for 25 min and centrifuged to remove cell debris. 10% of the total chromatin was processed for input and chromatin shearing was verified on a bioanalyzer. Chromatin extract was used to set up three IPs and one no antibody control per replicate and incubated with MYC antibody (BioLegend 626802) at 4°C overnight followed by 4hrs at 4°C with Protein G Dynabeads (Invitrogen 100-04D) pre-washed in FA-Lysis buffer. Beads were washed at room temperature with the following sequence of buffers; FA-Lysis buffer, two washes in FA-Lysis buffer with 500 mM NaCl, TEL buffer (0.25M LiCl, 10 mM Tris–HCl pH 8.0, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate), twice with 1XTE. Dynabeads from the three IPs per replicate and three no antibody controls (one from each replicate) were combined during the final wash. Chromatin fragments were eluted using 200 μl of Elution Buffer (TE + 1% SDS) at 65°C 20 min with agitation. NaCl was added to a final concentration of 250mM, and crosslinks were reversed overnight at 65°C. Elutions were treated with RNAse 1hr at 37°C and proteinase K for 2hr at 55°C. DNA was purified by running the elutions through a Qiagen PCR purification column. Libraries were prepared with a Sciclone liquid-handling robot using the KAPA HTP Library Prep Kit for Illumina (Roche KK8234) and Bio Scientific NEXTflex DNA barcodes (PerkinElmer). NEXTflex DNA barcode adapters were used at a dilution of 1:125 with 15 cycles of PCR. The resulting libraries were then quantified using a Bioanalyzer (Agilent Technologies) and a Qubit fluorometer (Life Technologies). Libraries were pooled and sequenced on an Illumina NextSeq 500 instrument as High Output 75 bp single read runs.