title: GSM8656967: SEM MLLT3; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP-seq Briefly, 10^7 cells were single-fixed (1% formaldehyde for 10 min) for histone modifications or double-fixed (2 mM disuccinimidyl glutarate for 30 min, then 1% formaldehyde for 30 min) for transcription/chromatin factors, then lysed with 120 µl SDS lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS) and sonicated using a Covaris ME220 (Woburn, MA) to generate 200–300 bp fragments. Insoluble material was pelleted, then the supernatant was diluted 10× and pre-cleared for 30 min at 4 °C with rotation using 5 µl protein A- and G-coupled dynabeads (ThermoFisher Scientific). An input sample was taken, then 2 µg antibody was added to the sample before incubation overnight at 4 °C with rotation. The antibodies used are detailed in Supplementary Table 3. Protein A- and G-coupled dynabeads were used to isolate antibody-chromatin complexes, after which the beads were washed three times with RIPA buffer (50 mM HEPES-KOH pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP40 and 0.7% sodium deoxycholate) and once with Tris-EDTA. Samples were eluted with SDS lysis buffer, RNase A- and proteinase K-treated and crosslinks were reversed at 65 °C overnight. DNA was purified by PCR purification column (Qiagen) and analyzed by qPCR, relative to input. PCR primer sequences are given in Supplementary Table 4. For ChIP-seq, libraries were generated using the Ultra II library preparation kit (NEB), then sequenced by paired-end sequencing with a 75-cycle high-output Nextseq 500 kit (Illumina).