GSM2228956: CS: STAT5A ChIP HODHBt/IL-2; Homo sapiens; ChIP-Seq
Cells were crosslinked by adding 16% paraformaldehyde to a final concentration of 1% in media. Crosslinking was allowed to proceed for 15 min before Tris pH 7.6 addition to 1.33 M. Cells were pelleted, rinsed twice in PBS, decanted, and frozen at -80. Pellets containing 1.2 million cells were thawed and Dounce homogenized in 1 ml 0.2% Sarkosyl Buffer (20 mM Tris pH 7.6, 150 mM NaCl, 1 mM EDTA, 0.2% Sarkosyl), then sonicated, after which 100 microliters of 10% Triton X-100 was added. Sonicated samples were pelleted at 16000 x g for 15 min and supernatants were precleared with 50 microliters PBS-washed Protein A Dynabeads (Life Technologies 10001D) for 3 h at 4 degrees. In parallel, 50 microliters Protein A Dynabeads were PBS-washed, blocked in 50 microliters PBS containing 1 mg/ml BSA for 1 h at 4 degrees, washed again with PBS, and incubated with 15 micrograms anti-STAT5A (sc-1081). Antibody-beads were washed twice with 100 microliters Wash Buffer A (20 mM Tris pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS), then resuspended with precleared sample and incubated overnight at 4 degrees. 1% of each precleared sample was set aside for input sequencing. Samples were washed once with 200 microliters Wash Buffer A, 200 microliters Wash Buffer B (20 mM Tris pH 7.6, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS), and 200 microliters Wash Buffer C (20 mM Tris pH 7.6, 250 mM LiCl, 1 mM EDTA, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, and 0.1% SDS), then twice with 200 microliters Rinse Buffer (20 mM Tris pH 7.6, 50 mM NaCl, 1 mM EDTA). Beads were incubated with 200 microliters Elution Buffer (20 mM Tris pH 7.6, 1 mM EDTA, 1% SDS) for 2 h at 65 degrees to reverse crosslinks. Eluates were treated with 2 microliters RNase A for 30 min at 37 degrees, then 2 microliters Proteinase K for 30 min at 50 degrees. DNA was isolated using a MinElute PCR Purification Kit (QIAGEN 28004). Libraries were prepared using a NEXTflex Rapid DNA-Seq Kit (Bioo Scientific 5144-03)
GEO Accession
GSM2228956