title: GSM8686511: MUTZ-3 dTAGV-1 6hr H3K27ac ChIP; Homo sapiens; ChIP-Seq
library_name: GSM8686511
library_construction_protocol: Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed on chromatin from 1×106 CD34+ MUTZ-3-dTAG cells after treatment with 500nM dTAGV-1 or DMSO for 6 hours. Cells were cross-linked with 1% methanol-free formaldehyde (Pierce Life Technologies, 28906), quenched with 0.125 M glycine and frozen at −80 °C and stored until further processing. ChIP reaction was performed with iDeal ChIP-seq kit for TFs (Diagenode, C01010055) with modifications of the manual detailed below. Lysed samples were sonicated using the E220 sonicator (Covaris, 500239) in microTUBE AFA Fiber Pre-Slit Snap-Cap tubes (Covaris, 520045) with settings for 200-bp DNA shearing. Sheared chromatin was immunoprecipitated with 2.5 μg HA antibody (CST, HA-Tag (C29F4) Rabbit mAb #3724), 2.5 μg H3K27ac antibody (Diagenode, C15410196) or 2.5 μg IgG antibody (Diagenode, C15410206, RRID AB_2722554). Eluted and decross-linked DNA was purified with MicroChIP DiaPure columns (Diagenode, C03040001) and eluted in 30 μl of nuclease-free water. ChIP and input libraries for sequencing were prepared with ThruPLEX DNA-Seq kit (Takara, R400674) and DNA Single Index kit, 12S Set A (Takara, R400695). Size selection steps were performed with Magbio Genomics HighPrep PCR beads (Fisher Scientific, 50-165-6582). The libraries were sequenced using the Illumina NextSeq 550 platform and the 150-bp paired-end configuration to obtain at least 20 million reads per sample.