HEK 293T cells CRISPR-Cas9 pull-down

HEK 293T cells were transfected at 60–80% confluence in 10 cm plates using the jetOPTIMUS transfection kit (Polyplus Transfection) with either pCDNA3.1-V5-dSpCas9, pCDNA3.1-3xFLAG-dSpCas9, or pCDNA3.1(-). Forty-eight hours post-transfection, biological replicates of confluent 10 cm plates of HEK 293T cells were treated with 400 mJ/cm² of UV using the Stratalinker 2400, harvested in ice cold PBS and pellets flash frozen in liquid nitrogen and stored in −80 °C until ready to IP with either V5 Tag mouse monoclonal antibody (ThermoFisher Scientific #R960-25) or mouse monoclonal ANTI-FLAG M2 antibody (Sigma-Aldrich #F1804) each at a dilution of 1:3000 in a subsequent protocol exactly as detailed in Van Nostrand et al.^{6 (link)}, cutting the nitrocellulose membrane from 115 kDa and up. The size-matched input not subjected to IP was cut from the identical region. Sequencing was performed on Illumina HiSeq 4000 with paired end reads.