GSM2486815: HUVEC RAD21 ChIP-seq hypoxia replicate 1; Homo sapiens; ChIP-Seq
Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with antibody. Libraries were prepared according to Illumina's instructions. 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 (minus exo) 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, ChIP DNA was PCR-amplified with NEXTflex DNA barcodes and library fragments were size-selected (230-350bp) from a 10% TBE-gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on Illumina HiSeq 2000 according to the manufacturer’s instructions.