title: GSM9165010: K562-CTCF_MO-ChIP-exo_(10_cycles)_rep1_262-111; Homo sapiens; OTHER
library_construction_protocol: K562 suspension cells were counted and directly crosslinked in media or pelleted and resuspended for crosslinking in PBS. Adherent cells were washed with PBS, trypsinized, quenched and resuspended in media or PBS for counting and crosslinking. All cell counts were performed with a trypan blue dye exclusion test using a hemocytometer. For MO-ChIP-exo, 10 min of crosslinking with 1% formaldehyde was quenched with a 3M Tris pH 7.8. Variations of the crosslinking time, formaldehyde concentration and quencher were used as reported. Crosslinked cells were washed with PBS + 1X CPI and flash frozen in liquid nitrogen. Crosslinked cell pellets (10 M cells per reaction) were subjected to cell and nuclear lysis. Lysates were resuspended in PBS + 1X CPI for sonication in a Diagenode Bioruptor Pico. The 'Go & Shear' setting was used with the validated parameters on the 'Easy Mode' for 30''ON/OF with increasing cycles (1-11 as described). An aliquot of 15 μL (~0.5 M cells) was used to reverse crosslink and purify the DNA for a sonication assessment using the Agilent Tape Station D5000 kit. The sonicated crosslinked chromatin was pre-cleared before ChIP using high speed centrifugation (14k rpm at 4C for 15 min). ChIP antibodies [Anti-CTCF (Sigma 07-729), Anti-USF1-1B8 (DSHB USF1-1B8-6/13/19), IgG (Sigma i5006), Anti-GATA1 (Santa Cruz Biotechnology  SC-266), Anti-FoxA1 (Santa Cruz Biotechnology SC-101058), Anti-SOX2 (R&D AF2018)] were pre-conjugated to 50 μL Protein A or G Dynabeads (Invitrogen) before an overnight incubation with the antibody of interest at 4C. Bead-bound chromatin was washed and subjected to simultaneous End-repair and A-tailing with a subsequent TA ligation of the first HPLC-purified adapter using the NEBNext® Ultra™ II DNA Library Prep Kit. After washes, a fill-in reaction was performed using Phi29 DNA Polymerase (NEB). This reaction was followed by another wash and Lambda Exonuclease (NEB) digestion. Reverse crosslinking was performed overnight to release the DNA. Released DNA was purified using Mag-Bind® TotalPure NGS beads (Omega Bio-Tek) before ligating the 2nd HPLC-purified adapter using T4 DNA ligase (Qiagen). A DNA cleanup was performed using Mag-Bind® TotalPure NGS beads before PCR enrichment for 12 cycles. This was followed by size selection using SPRI select beads (Beckman Coulter) and an additional DNA cleanup to remove excess adapters. Completed libraries were analyzed in the Tape Station using High Sensitivity D5000 ScreenTape and Reagents. Finally, libraries were individually quantified through qPCR using NEBNext® Library Quantification Kit, For Illumina® (NEB) in an ABI StepOne Plus Real Time PCR System. Completed libraries were pooled for each sequencing run based on their concentration and read allocation with a 1% PhiX spike-in, treated with Illumina's Free Adapter Blocking Reagent and cleaned up using Mag-Bind® TotalPure NGS beads. Final pool concentration was determined through qPCR using NEB Next® Library Quantification Kit, For Illumina® in an ABI StepOne Plus Real Time PCR System before diluting to 0.65 nM for loading. An Illumina NextSeq 2000 Sequencing System was used with a NextSeq™ 1000/2000 P2 XLEAP-SBS™ Reagent Kit (100 Cycle) for paired-end sequencing with 50 bp each for read1 and read2.