title: GSM3182663: MCF10AsiSIER_4OHT2h_XRCC4_chip; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP-seq: Cells were cross-linked by adding formaldehyde to the culture medium to a final concentration of 1% (8 min at RT). Cross-linking was stopped by addition of glycine to a final concentration of 125 mM. Cells were washed twice with PBS and lysed in SDS buffer: 100 mM NaCl, 50 mM Tris HCl (pH 8.1), 5 mM EDTA (pH 8), 0.5% SDS, and protease inhibitors. Chromatin lysate was then pelleted and resuspended in IP buffer (100 mM NaCl, 100 mM Tris HCl at pH 8.1, 5 mM EDTA at pH 8, 0.3% SDS, 1.7% Triton X-100), where it was directly sonicated prior to overnight incubation with antibodies . ChIP-seq: Libraries were prepared according to HT-ChIP-Seq Library Preparation Protocol. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3´→ 5´ exo minus) and dATP to yield a protruding 3- 'A' base for ligation of indexed oligo adaptors which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with amplification primers for 14 cycles. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.