title: GSM6045991: MOLM13_dIRF8_4h_DMSO_PU1_rep3_S14; Homo sapiens; ChIP-Seq
library_construction_protocol: For all the experiments, cells were collected in culture media, and crosslinked in 1% formaldehyde for 20 min at room temperature, and the reaction was sequestered with 0.125M Glycine in PBS for 10 min. The crosslinked cells were spun down, washed with PBS and freezed in -80°C until use. Ten million cells were used for H3K27ac and H3K4me1, and 50 million cells for the SPI1, respectively. Cell pellets were lysed in cell lysis buffer (10mM Tris pH 8.0, 10 mM NaCl, 0.2% NP-40) with protease inhibitor (Roche) and incubated for 15 min on ice. The cells were spun down for 30 seconds at 4,200 RPM and the buffer was removed. The cells were resuspended in 1ml nuclear lysis buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS) per 10 million cells, followed by a 10 min incubation on ice. The cells were sonicated with a bioruptor for 5 mins at 4°C and spun down at max speed for 15 mins at 4°C. Supernatants containing fragmented chromatins were collected, diluted with 7ml IP dilution buffer (20 mM Tris pH 8.0, 2 mM EDTA, 50 mM NaCl, 1% Triton X-100, 0.01% SDS) per 10 million cells, and applied with respective antibodies (PU.1: 10 μg, CST#2266; H3K27ac: 2 μg, #ab4729, H3K4me1: 2μg, #ab8895). For each antibody, 30 μl of protein A magnetic beads (Invitrogen) were applied after washing with 0.5% BSA in TBS for 3 times and blocked at 4°C on a rotor for 2h. The mixture was incubated overnight at 4°C in rotation. The beads were washed once with IP wash I buffer (20 mM Tris pH 8.0, 2 mM EDTA, 50 mM NaCl, 1% Triton X-100, 0.01% SDS), twice with high salt buffer (20 mM Tris pH 8.0, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.01% SDS), once with IP wash 2 buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate), and twice with 1xTE. The beads were incubated with 200 μl elution buffer (50mM Tris 8.0,10mM EDTA, 1%SDS), vortexed 3x for 5s, followed by shaking at 950 RPM at 65°C for 15 mins. Supernatants were collected, and reverse crosslinking was performed in 0.25M NaClat 65°C overnight. non-DNA content was eliminated with 2 μl RNase A for 1 hr at 37 °C, followed by 0.2 mg/ml Proteinase K for 2 hr at 42°C. ChIP DNA was purified using QIAquick PCR purification kit according to manufacturer's instructions, and eluted with 40 μl water. ChIP-seq libraries were constructed using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (E7645L), and the optimal cycle numbers were determined by NEBNext® Library Quant Kit for Illumina (E7630L). Library quality was assessed using the high sensitivity Bioanalyzer kit (Agilent) and libraries were pooled and single-end sequenced for 75 bp on the Nextseq 500 platform.