title: GSM4669249: ChIP_Pano_IFNLPS_1; Homo sapiens; ChIP-Seq
library_construction_protocol: We used 4x106 cells per sample from 6 well dishes for H3K27ac ChIP-Seq (n=2/group). During cell harvesting, cell culture medium was removed and cells were treated with PBS+1% PFA (Amresco, M134) at RT for 15 minutes and then quenched with 0.125 M glycine at RT for 5 minutes. After washing 6 times with cold PBS, the nuclei were extracted by incubating cells in RLB buffer (50 mM HEPES pH7.9, 140 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, and cOmplete ULTRA Protease Inhibitor Cocktail, Sigma 582988001) on ice for 1 hour. After washing with MNase buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1% Triton X-100, 1 mM CaCl2, 1 mM DTT), chromatins were digested with 66 units MNase (New England Biolabs, M0247S) at 37 oC for 10 minutes and the reactions were stopped using EGTA (5   M) on ice for 5 minutes. Chromatins were sonicated with a Branson Microtip Sonifier 450 (4x15 second at output 4.5 and duty cycle 60%). The sizes of the chromatins after these treatments were ~150-500 bp in length. For ChIP, we incubated the chromatins with 20   l Dynabead protein G (Thermo Fisher Scientific, 10004D) and 1.5   g anti-H3K27ac antibodies (Abcam Cat# ab4729, RRID:AB_2118291) in IP dilution buffer (20 mM Tris HCl pH8.0, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA) at 4 oC overnight. IPed chromatins were sequentially washed with IP dilution buffer+0.1% Triton, ChIP wash I (20 mM Tris HCl pH8.0, 150 mM NaCl, 0.5% Triton, 2 mM EDTA, 2 mM EGTA), ChIP wash II (20 mM Tris HCl pH8.0,  500 mM NaCl, 1% Triton, 2 mM EDTA, 2 mM EGTA), ChIP wash III (20 mM Tris HCl pH8.0, 250 mM LiCl, 1% Triton, 2 mM EDTA, 2 mM EGTA) and TE buffer. Chromatins were eluted using elution buffer (20 mM Tris HCl pH8.0, 5 mM EDTA, 5 mM EGTA, 1% SDS) at 65oC for 2 x 15 minutes. Chromatins were digested using RNase ONE Ribonuclease (Promega, PAM4261, 1:10 dilution) at 37oC for 1 hour and Pronase (Roche, 10165921001, 1.5   g/  l) at 42oC for 2 hours and 65oC for 6 hours. Digested chromatins were purified using Qiagen PCR purification kits. ChIP-Seq libraries were prepared using a standard protocol, including End-Repair, dA-Tailing, Single-Index Adapter Ligation and final PCR Amplification (17 cycles) using Phusion DNA polymerase. ChIP-Seq libraries were purified from 2% agarose gels. Concentrations of ChIP-Seq libraries were determined using a Bioanalyzer. ChIP-Seq libraries were sequenced using a NextSeq 500/550 High Output V2.5 75 cycles kit from Illumina on a Nextseq 550 platform and all data were deposited to GEO. For normalization controls, 1% of the chromatin that was used for ChIP was used to make input libraries.
attributes.tag: GEO Accession
attributes.value: GSM4669249