4ug each of Anti-Bcl11b Antibodies: Abcam ab18465, Bethyl A300-385, and 20ul of CST D6F1
Sequenced DNA Library
Isolated cells were fixed with 1% formaldehyde in PBS for 10 minutes, then quenched with glycine. After washing, DNA was extracted using the Illumina SimpleChIP Enzymatic ChIP kit (#9003S). Briefly, nuclei were isolated by incubating in 1ml buffer A for 10 min on Ice, followed by centrifugation and resuspension in 1 ml Buffer B. Chromatin was sheared to roughly 200-1000bp. Samples were centrifuged to clear lysates, and the supernatant containing the DNA was transferred to a fresh tube for overnight antibody complexing. Next day, protein G dynabeads were added to each tube, incubated, washed, and isolated with magnet. DNA complexes were then released from the beads, decrosslinked, and phenol:choloroform:isolamyl alcohol extracted for library prep. DNA libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina. Briefly, DNA ends were repaired, followed by ligation of adapter sequences. DNA was cleaned with AMPure XP beads, and then DNA was PCR amplified with 7-8 cycles. After final cleaup with AMPure XP Beads, libraries were quantified using Agilent Bioanalyzer high sensitivity DNA kit and were subsequently sequenced.