Affinity-seq for Identifying Protein-DNA Interactions

Affinity-seq was essentially done as in (24 (link)) with minor adjustments. A ZF array of the protein of interest was amplified then cloned into a universal Affinity-seq vector by recombineering. The resulting construct expressed a fused protein containing 6HisHALO–the 412–511 aa fragment of PRDM9–ZF array of interest. The fused protein was expressed in Rosetta 2 cells at 15°C for 24 h and partially purified by ion exchange chromatography on SP-sepharose. The purified protein was mixed with genomic DNA sheared to ∼200 bp on a Covaris ultrasonicator, and allowed to bind overnight. The protein-DNA complexes were then isolated on HisPur Ni-NTA Resin (Thermo Scientific) preincubated with a partially purified prep of the empty tag to reduce the background. DNA was then eluted and used to prepare genomic libraries using a TruSeq ChIP Library Prep Kit (Illumina). The libraries were sequenced on a HiSeq2500 or NextSeq platform ensuring ∼50 million reads per library. Data were analyzed using a custom pipeline as described previously (24 (link)) and we used the MEME (v4.10.1) software package with default parameters (P-value threshold 0.001 and 150 bp central peaks regions) for motif discovery.