title: GSM6255144: DIPG-IV_ChIP_H3K27ac_Rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: For histone marks profiling, cells were crosslinked with 1 ml of 1% formaldehyde for 10 minutes. For CTCF and HA profiling cells were crosslinked in two steps with 2 mM of disuccinimidyl glutarate (DSG, Pierce) for 45 minutes at room temperature followed by 1 ml of 1% formaldehyde for 10 minutes. Crosslinked cells were then quenched with 0.125 M glycine for 5 minutes at room temperature and washed with PBS. After fixation, pellets were resuspended in 500 μl of 1% SDS (50 mM Tris-HCl pH 8, 10 mM EDTA) and sonicated for 5 minutes (histone marks)  or 10 minutes (HA and CTCF) using a Covaris E220 instrument (setting: 140 peak incident power, 5% duty factor and 200 cycles per burst) in 1 ml AFA fiber millitubes. Chromatin was immunoprecipitated with 10 μg of each antibody (H3K4me3 Abcam ab8580, RRID:AB_306649; H3K27ac Diagenode C15410194, RRID:AB_2637079; CTCF Millipore 07-729, RRID:AB_441965; HA Abcam ab9110, RRID:AB_307019). 5 μg of chromatin was used for histone mark ChIPs, and 40 μg of chromatin was used for HA and CTCF ChIPs. ChIP-seq libraries were made using NEBNext® Ultra™ II DNA Library Prep Kit (New England BioLabs, E7103). 75 bp paired-end reads were sequenced on a NextSeq instrument (Illumina).