HIRA (approximately equimolar mixture of WC15, WC19, WC117, WC 119)
Sequenced DNA Library
Cross-linked cells were solubilized by ultrasound treatment (Bioruptor Diagenode) to generate DNA fragmentation in 150-300 bp range. Protein-DNA complexes were isolated using corresponding antibody pre-immobilized on Dynabeads (Invitrogen). Libraries were prepared according to NEB's instructions accompanying the NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina®(E6240S). Briefly, DNA was end-repaired using a combination of T4 DNA 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 library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were isolated using Agencourt AMPure XP beads (Beckman Coulter) and PCR-amplified using Illumina primers. 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.