GSM2976468: ChIP_NPC_Mkl1OE_input; Mus musculus; ChIP-Seq
Cells were fixed in 1% paraformaldehyde at room temperature for 10 min followed by quenching with 125 mM glycine at room temperature for 5 min. Fixed cells were washed with cold PBS twice. Cells were harvested by scraper and centrifuged at 700 × g for 3 min and then resuspended in nucleic lysis buffer (10 mM Tris-HCl, 10mM EDTA, 1% SDS, 200 mM NaCl, pH 7.5). The genomic DNA was fragmented with Bioruptor (Cosmo Bio) at 4 °C to a size range between 300 and 600 bp. Cell contaminants were removed by centrifugation. A part of cleared solution was used as the input sample. An anti-SRF antibody (clone D71A9; Cell Signaling Technology) or anti- Histone H3 acetyl Lys27 antibody (Active Motif, #39133) were prebound by incubating with Protein-G Dynabeads (Thermo Fisher Scientific) in IP dilution buffer (16.7 mM Tris-HCl, 1.2 mM EDTA, 1.1% (vol/vol) Triton X-100, 167 mM NaCl, 0.01% SDS, pH 8.0) and then incubated at 4 °C overnight with constant rotation. Samples were washed twice with IP dilution buffer, twice with low NaCl buffer (20 mM Tris-HCl, 2 mM EDTA, 1% Trion X-100, 150 mM NaCl, 0.1% SDS, pH 8.0), twice with high NaCl buffer (20 mM Tris-HCl, 2 mM EDTA, 1% Trion X-100, 500 mM NaCl, 0.1% SDS, pH 8.0), twice with LiCl buffer (10 mM Tris-HCl, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, pH 8.0) and twice with TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The DNA-protein complex was finally eluted with elution buffer (25 mM Tris-HCl, 5 mM EDTA, 0.5% SDS, pH 7.5). Eluates and input samples were treated with Pronase (Roche) at 42 °C for 2 h followed by 65 °C overnight with constant shaking. DNA was purified with MinElute Reaction Cleanup Kit (Qiagen). Immunoprecipitated DNA was ligated with adaptors of Illumina TruSeq using TruSeq ChIP Sample Prep Kit (Illumina). Libraries were assessed for quality and quantity using Agilent Bioanalyzer 2000 and the KAPA Library Quantification Kit for Illumina (KAPA Biosystems).
GEO
GSM2976468