GSM6294389
Genomic DNA (input) was prepared for asynchronous and mitotic K562 cells by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by SPRI beads clean up (Beckman Coulter) and quantitation by Clariostar (BMG Labtech). 40 µg of chromatin was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 5 µg of antibody against GATA2 (Santa Cruz, sc-9008). Complexes were washed, eluted from the beads with SDS buffer, and treated with RNase and proteinase K. Crosslinks were reversed by incubation overnight at 65 ºC, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Quantitative PCR (qPCR) reactions were carried out in triplicate on specific genomic regions using SYBR Green Supermix (BioRad). The resulting signals were normalized for primer efficiency by carrying out qPCR for each primer pair using Input DNA. Illumina sequencing libraries were prepared from ChIP and Input DNAs by standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. Steps were performed on an automated system (Apollo 342, Wafergen Biosystems/Takara). After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on Illumina's NextSeq 500 (75 nt reads, single end)