title: GSM3498572: shControl_SMC1a; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were trypsinized, harvested and the cell number was estimated. Cross-linking was performed using 1 percent formaldehyde for 10 min at room temperature followed by quenching with 0.125 M Glycine. Cross-linked pellets were washed twice with ice-cold 1X PBS (with 0.1 percent Triton X-100) and then subjected to lysis with a lysis buffer (10 mM Tris-Cl (pH 8), 100 mM NaCl, 10 mM EDTA, 0.25 percent Triton X-100 and protease inhibitor cocktail (Roche)). Pellets were resuspended after centrifugation, in 1 percent SDS lysis buffer (50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 1 percent SDS, 2 mM EDTA, 1 percent Triton X-100, 0.1 percent NaDOC and protease inhibitor cocktail). For every 10 million cells, 14 cycles of sonication were carried out using the Bioruptor (Diagenode) with 30 sec pulses and 60 sec halts in each cycle. Pre-clearing of the chromatin was carried out with 100 uL of Protein G Dynabeads (Life Technologies) for 2 hours at 4 degree Celsius. Simultaneously, 100 uL of Protein G Dynabeads were also bound to 5 microgram (per 10 million cells) of the antibodies (Setdb1 – home-made Ab; Setdb1 - Santa Cruz #sc-66884; Smc1a - Bethyl #A300-055A; Flag - Sigma-Aldrich #F1804; Pan Methyl Lysine - Biorbyt #orb100017). After separation of a small amount as input - the remaining pre-cleared chromatin was used to bind to the antibody-bound beads at 4 degree Celsius, overnight. Elution involved three washes with 0.1 percent SDS lysis buffer, one with 0.1 percent SDS lysis buffer/0.35 M NaCl, one with 10 mM Tris-Cl (pH 8), 1 mM EDTA, 0.5 percent NP40, 0.25 LiCl, 0.5 percent NaDOC, and one with TE buffer (pH 8.0). The immunoprecipitated chromatin was eluted out from the beads by heating the beads resuspended in 50 mM Tris-HCl (pH 7.5), 10 mM EDTA, 1 percent SDS, for 1 hour at 68 degree Celsius while shaking at 1400 rpm. Cross-links were reversed by incubating the eluted samples and inputs at 42 degree Celsius for 2 hours and 67 degree Celsius for 6 hours in the presence of Pronase (Sigma) and TE buffer, after which the DNA was purified using the QIAGEN PCR Purification kit (for inputs) and the QIAGEN MinELute PCR Purification kit (for samples). Following qPCR validation of the samples, libraries were prepared by using Illumina reagents with TruSeq adapters and the TruSeq ChIP Library Preparation kit, as per the manufacturer's instructions. The quality and concentration of each sample was assessed by using Agilent High Sensitivity chips on the Agilent 2100 Bioanalyzer.