title: GSM1174239: Tumors IL6 (CisGenome); Mus musculus; ChIP-Seq
library_construction_protocol: Following DNA-Protein cross-linking with formaldehyde and tissue homogenization, chromatin was extracted using "shearing buffer" provided in the SIGMA Imprint ChIP Kit in accordance with the manufacturer's instructions. Chromatin shearing was performed using the Diagenode Bioruptor water bath sonicator. Sheared chromatin was used for immunoprecipitations with a ChIP-validated Stat3 antibody (Santa Cruz SC-482X) and the SIGMA Imprint ChIP Kit according to the manufacturer's instructions. Libraries were prepared according to Illumina's instructions accompanying the ChIP-Seq DNA Sample Preparation Kit (IP-102-1001). 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 Exo fragment 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. Following adapter ligation, DNA was PCR amplified (15 cycles) with Illumina primers, and library fragments of 200-300 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 II following the manufacturer's protocols.