title: GSM4200196: Hmga2+/+ TGFB1 pH2AX input; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were cross-linked with 1% formaldehyde for 10 min at room temperature. The reaction was quenched for 5 min on ice by adding 0.125 M glycine. After three washes with 1x PBS, cells were subjected to hypotonic buffer L1 (50 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0, 0.1% IGEPAL CA-630 (Sigma), 10% glycerol, 2 mM DTT, 10 mM NaF, 2 mM Na3VO4, 0.2 M imidazole, 1 mM PMSF and protease inhibitor cocktail). Cells were lysed, and sonicated with Diagenode Bioruptor to an average DNA length of 100-300 bp. After centrifugation, the soluble chromatin was immunoprecipitated using antibodies specific against p H2A.X (Millipore), SUPT16H (CellSignaling) and SSRP1 (BioLegend). Precipitated chromatin complexes were removed from the beads and decrosslinked by incubating with 100 μl of 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.1 M NaHCO3, 1% SDS and Proteinase K at 65 °C overnight. Reverse cross-linked immunoprecipitated chromatin was purified using the QIAquick PCR purification kit (Qiagen). Precipitated DNA samples were purified by QIAquick PCR purification kit (Qiagen) and quantified by Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). 2ng of DNA was used as input for TruSeq ChIP Library Preparation Kit (Illumina) with following modifications. Instead of gel-based size selection before final PCR step, libraries were size selected by SPRI-bead based approach after final PCR with 18 cycles. In detail, samples were 1st cleaned up by 1x bead:DNA ratio to eliminate residuals from PCR reaction, followed by 2-sided-bead cleanup step with initially 0.6x bead:DNA ratio to exclude larger fragments. Supernatant was transferred to new tube and incubated with additional beads in 0.2x bead:DNA ratio for eliminating smaller fragments, like adapter and primer dimers. Bound DNA samples were washed with 80% ethanol, dried and resupended in TE buffer. Library integrity was verified with LabChip Gx Touch 24 (Perkin Elmer).
attributes.tag: GEO Accession
attributes.value: GSM4200196