GSM6605301: MUTZ-3 MECOM edited  MECOM_1; Homo sapiens; ChIP-Seq
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed on chromatin from 2e6 MUTZ-3 MECOM and AAVS1 edited cells after magnetic sorting for CD34+ cells (Stemcell 17856). Sorted 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 Transcription Factors (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 200bp DNA shearing. Sheared chromatin was immunoprecipitated with 2.5μg CTCF antibody (abcam, ab128873, RRID:AB_11144295) or 2.5μg IgG antibody (Diagenode, C15410206, RRID: AB_2722554). Eluted and decross-linked DNA was purified with MicroChIP DiaPure columns (Diagenode, C03040001) and eluted in 30μL of NucleaseFree 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 Inc HighPrep PCR beads (Fisher Scientific, 50-165-6582). Subsequently, ChIP-seq library was quantified with Agilent Bioanalyzer. The libraries were sequenced at Broad Institute Genomic Services by using the Illumina NextSeq 500 platform and the 150-bp paired-end configuration to obtain at least 30 million reads per sample.