DNA–RNA immunoprecipitation sequencing (DRIP-seq)

To perform DRIP-seq⁷⁰, cells (5 × 10⁶) were lysed in 0.5% SDS/TE, pH 8.0 containing Proteinase K overnight at 37 °C. Total DNA was isolated with phenol/chloroform/isoamylalcohol extraction followed by standard ethanol precipitation. One-third of total DNA was fragmented by a cocktail of restriction enzymes (EcoRI, HindIII, BsrgI, SspI, XbaI) overnight at 37 °C. A negative control treated overnight with RNase H was included. Digested DNA was purified by phenol/chloroform/isoamylalcohol extraction, ethanol precipitation and quantified by Nanodrop. Four micrograms of digested DNA were diluted in binding buffer (10 mM NaPO₄, pH 7.0; 0.14 M NaCl; 0.05% Triton X-100) and incubated with 10 μg of S9.6 antibody overnight at 4 °C on a rotator. DNA/antibody complexes were added for 2 h at 4 °C to Agarose Protein-A/G beads prewashed with binding buffer. Immunoprecipitated DNA was eluted by incubating with elution buffer (50 mM Tris pH 8.0; 10 mM EDTA; 0.5% SDS) containing Proteinase K at 55 °C for 45 min on a rotator. The eluent was precipitated by phenol/chloroform/isoamylalcohol extraction and ethanol precipitation. Validation of DRIP procedure was performed by qPCR (see Supplementary Table ² for primer sequences). The pulled down material and input DNA were then sonicated, size-selected, and ligated to Illumina barcoded adaptors, using TruSeq ChIP Sample Preparation Kit (Illumina) or ThruPLEX® DNA-seq Kit (Rubicon Genomics) for next-generation sequencing (NGS) on Illumina HiSeq 2500 platform.