title: GSM2027838: Sir3∆wH 2µ (CL1+pCL4); Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: Cell pellets were resuspended in 600 μl lysis buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate, 0.1% SDS, 1 mM PMSF, protease inhibitor tablet (Roche)), and disrupted by bead beating (MagNA Lyser, Roche) for 6x30 sec at 4500 rpm with 0.5 mm glass beads. Tubes were punctured and the flow-through was collected in a new tube by centrifugation. After sonication for 3x20 sec at 40% amplitude (Branson Digital Sonifier), the extract was centrifuged (Eppendorf 5415R) for 15 min at 13,000 rpm. The soluble chromatin was then transferred to a fresh tube. Sir3 antibody (Rudner et al., 2005) was preincubated with Dynabeads Protein A, and for each immunoprecipitation, 2 μg antibody bound to 30 μl beads was added to soluble chromatin. Samples were incubated for 2 hours at 4oC with rotation, after which the beads were collected on magnetic stands, and washed 3 times with 1 ml lysis buffer and once with 1 ml TE, and eluted with 100 μl preheated buffer (50 mM Tris.HCl pH 8.0, 10 mM EDTA, 1% SDS) at 65 ˚C for 15 min. The eluate was collected, and 150 μl 1XTE/0.67% SDS was added. Immunoprecipitated samples were incubated overnight at 65 ˚C to reverse crosslink, and treated with 50 μg RNase A at 37oC for 1 h. 5 μl proteinase K (Roche) was added and incubation was continued at 55oC for 1 h. Samples were purified using PCR purification kit (Qiagen). Libraries for Illumina sequencing were constructed following the protocol detailed in Wong et al, 2013 (Wong, K. H., Jin, Y. and Moqtaderi, Z. 2013. Multiplex Illumina Sequencing Using DNA Barcoding. Current Protocols in Molecular Biology. 101:7.11:7.11.1–7.11.11.), starting with ~5 ng of immunoprecipitated DNA fragments. Each library was generated with custom-made adapters carrying unique 6 nt barcode sequences at the ligating end. Barcoded libraries were mixed and sequenced with Illumina HiSeq 2500.