GSM1542194: PolII_ChIPSeq, TPA-treated HL-60 cells; Homo sapiens; ChIP-Seq
HL-60 cells were cultured for 48 h in the presence of 32 nM TPA or DMSO control as indicated and cross-linked by the addition of 1/10 volume of freshly-prepared formaldehyde cross-linking buffer (11% formaldehyde, 10 mM HEPES-KOH [pH 7.6], 100mM NaCl, 1 mM EDTA, 0.5 mM EGTA) to cell culture dishes and incubated for 10 min at room temperature. Glycine was added to a final concentration of 125 mM and cells were incubated for 5 min at room temperature. The samples were sonicated and the sonicated lysates were subjected to immunoprecipitation using the indicated antibodies and protein A/G Agarosebeads. Precipitates were washed and DNA was extracted. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer IIx following the manufacturer's protocols.