ChIP-seq and RIP-seq Library Preparation

Libraries for ChIP-seq were prepared according to manufacturer's instructions (Illumina, San Diego, CA) and as described (Asp et al., 2011 (link)). Briefly, IP'ed DNA (∼5 ng) was end-repaired using End-It Repair Kit (Epicenter, Madison, WI), tailed with deoxyadenine using Klenow exo⁻ (New England Biolabs), and ligated to custom adapters with LigaFast (Promega). Fragments of 300 ± 50 bp were size-selected and subjected to ligation-mediated PCR amplification using Phusion DNA polymerase (New England Biolabs). Libraries were quantified by qPCR using primers annealing to the adapter sequence and sequenced at a concentration of 7 pM on an Illumina Genome Analyzer IIx or 10 pM on an Illumina HiSeq. In some cases barcoding was utilized for multiplexing.
For RIP-seq libraries, polyA+ RNA was isolated using Dynabeads Oligo(dT)₂₅ (Invitrogen) beads and constructed into strand-specific libraries using the dUTP method (Parkhomchuk et al., 2009 (link)). Once dUTP-marked double-stranded cDNA was obtained, the remaining library construction steps followed the same protocol as described above for ChIP-seq libraries.