GSM3664675: DDX21 Chip on A77 1726  treated cells; Homo sapiens; ChIP-Seq
After chromatin immunoprecipitation, cross-link reversal is carried out and precipitated DNA is purified by phenol:chloform:isoamyl alcohol extraction. The End-It DNA End-Repair Kit (Epicentre) was used to turn DNA overhangs into phosphorylated blunt ends and the Agencourt AMPure XP PCR Purification Kit (Beckman Coulter) was used to purify the resulting samples using a 1.8X ratio of beads to sample. Next, a single A in the 3' end was added to samples using the Klenow Fragment enzyme (NEB) to allow for directional ligation and the AMPure Kit was again used to purify samples. Illumina adaptor oligos (1:10 dilution) were added to samples using the Quick Ligation Kit (NEB) and NEBNext Multiplex Oligos for Illumina Kit (NEB), and samples were purified with the AMPure Kit. Samples were then size-selected with the AMPure beads with a 0.9X bead to sample ratio. Multiplexing primers from the NEBNext Multiplex Oligos Kit were added during the 18-cycle PCR enrichment step, which utilized the Phusion High-Fidelity PCR Master Mix (NEB), to generate indexed libraries. Indexed libraries were beads purified. The concentration of the isolated libraries was estimated with a high-sensitivity DNA chip from Agilent according to manufacturer's protocol and libraries were mixed in equal quantities and sequenced on Illumina Hi-Seq2000. Index sequences from multiplexed primers were used to identify treatment group and reads were demultiplexed using a perl script.