GSM2661792: THP1 MΦ, GR, untreated, rep 2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
TFs and others
Cell type Class
Leukemia Acute Myelogenous
Attributes by original data submitter
Sequenced DNA Library
Cells were dual cross-linked in 2 mM disuccinimidyl glutarate solution followed by 1% methanol-free formaldehyde for 40 min total at room temperature and quenched in 0.125 M glycine for 10 min. Cells were then washed with PBS, scraped and lysed for 10 min at 4°C in lysis buffer (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) with protease inhibitor cocktail (Sigma) and the crude nuclear extract was collected by centrifugation at 600 g for 5 min at 4°C. Nuclei were washed for 10 min at 4°C in wash buffer (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0 with protease inhibitors) and collected as above. Nuclei were lysed (0.1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1 with protease inhibitors) and sonicated using a Bioruptor Pico (Diagenode) for THP1 M and hMΦ or an S220 Ultrasonicator (Covaris) for BMMΦ to generate DNA fragments approximately 150-600 bp. Inputs were taken from cleared lysates or incubated for 4 h at 4°C with the following antibodies to precipitate protein:DNA complexes: GR (Santa Cruz, M20x, sc-1004x or N499), GRIP1 (Abcam, ab10491 or Bethyl A300-346A), phospho-specific GRIP1 antibodies, or CDK9 (Santa Cruz, H169x, sc-8338x). 40-50 μl of pre-washed Dynabeads Protein A were added per IP and incubated O/N at 4°C. Beads were washed 8x in modified RIPA wash buffer (50 mM HEPES [pH 7.6], 100 mM LiCl [for H3Ac, H4Ac, H4K5/K8/K12Ac, BRD2/3/4 and P-TEFb (CDK9)] or 300 mM LiCl [H3, H3K4me3, Pol II and Pol II S2], 1mM EDTA, 1% NP-40 and 0.7% Nadeoxycholate) and 1x in TE containing 50 mM NaCl. Elution of DNA was performed in TE buffer containing 1% SDS After 6h cross-link reversal at 65 C, RNase digestion and Proteinase K digestion, ChIP DNA and input DNA were purified using the QIAGEN Quiaquick PCR purification kit. The amount and sonication quality of DNA was tested with Qubit (Thermo) and Bionalyzer 2100 (Agilent Technologies) Sequencing libraries were constructed using Illumina TruSeq ChIP-Seq Library Prep Kit and sequencing by a HiSeq 2500 system (4-6 samples/SR lane, 50 bp reads).