GSM7596744: PU.1 ChIP L2; Mus musculus; ChIP-Seq
Cells were cross-linked in 0.75% formaldehyde, pelleted, and frozen until the ChIP procedure. Pellets were thawed on ice, resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCL [pH 8.1] plus protease inhibitor) for 10 min. Volume was brought up to 1 ml with ChIP dilution buffer (16.7 mM Tris-HCl [pH 8.1], 167 mM NaCl, 0.01% SDS, 1.1% Triton X-100, and 1.2 mM EDTA plus protease inhibitor) and samples were sonicated with a Branson sonicator to shear chromatin. Chromatin was subsequently incubated with 1 µg PU.1 antibody (sc-390405; Santa Cruz Biotechnology) and rotated overnight at 4°C. Next, ChIPs were added to a 50:50 mix of prewashed Protein A/G Dynabeads (50 µl; 10001D/10009D; Thermo Fisher Scientific) and rotated overnight at 4°C. The next day, 175 µl RIPA/140 mM NaCl buffer (0.1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100,140 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl [pH 8.1]) was added to each tube and beads were transferred to a 96-well plate on a magnet, washed twice with cold RIPA/500 ml NaCl buffer (0.1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 140 mM NaCl, 1 mM EDTA, and 20 mM Tris-HCl [pH 8.1]), twice with cold LiCl buffer (0.25 M LiCl, 1% NP-40, 1% Na deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl [pH 8.1]), and twice with RT TE buffer (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA [pH 8.0]). Chromatin was eluted by adding 50 µl of elution buffer (10 mM Tris-Cl [pH 8.0], 5 mM EDTA, 300 mM NaCl, 0.1% SDS, and 5 mM dithiothreitol) and 8 µl of reverse cross-linking buffer (250 mM Tris-HCl [pH 6.5], 1.25 M NaCl, 62.5 mM EDTA, 5 mg/ml Proteinase K, and 62.5 μg/ml RNase A) and incubated at 65°C overnight. ChIP material was quantified on a Qubit spectrometer (Invitrogen) according to manufacturer's instructions. Library construction was performed first using an End-it DNA End-Repair Kit (ER0720; Epicenter) according to manufacturer's instructions, followed by DNA ligase-based adapter ligation (M2200S; New England Biolabs) and enrichment of adapter-modified DNA fragments by PCR. PCR fragments were gel purified using a Qiagen Gel Purification Kit (Qiagen) a PCR fragments were gel purified using a Qiagen Gel Purification Kit (Qiagen) and sequenced on an Illumina single-end flow cell. Sequencing of libraries was performed on Illumina HiSeq 2500v4 high-throughput sequencer.