GSM2024822: VCaP SOX9 ChIP-seq; Homo sapiens; ChIP-Seq
Cell was fixed by 1% formaldehyde for 10min and then lysed. Lysates were sonicated and Input DNA were set aside. Lysates were then clarified from sonicated nuclei and SOX9-DNA complexes were isolated with SOX9 antibody. Cross-linking of the DNA were reversed by heating for both Input and immunoprecipitated DNA. All DNA were purified by Qiagen PCR purification kit before library construction. Libraries were prepared according to Illumina's instructions accompanying the ChIP Sample Prep Kit. 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 following the manufacturer's protocols.