Jetoptimus transfection kit

HEK 293T cells were transfected at 60–80% confluence in Nunc Lab-Tek II Chamber Slides (ThermoFisher Scientific) using the jetOPTIMUS transfection kit (Polyplus Transfection) with either pCDNA3.1-V5-dSpCas9 or pCDNA3.1-3xFLAG-dSpCas9. Slides were fixed with MeOH, blocked for 1 h at room temperature, and incubated under gentle orbital shaking with primary antibody overnight: either V5 Tag mouse monoclonal antibody (ThermoFisher Scientific #R960-25) at 1:3000 dilution or mouse monoclonal ANTI-FLAG M2 antibody (Sigma-Aldrich #F1804) at 1:1000 dilution. Slides were washed five times for 10 min with phosphate-buffered saline with Tween 20 (PBST), then incubated for 1 h at room temperature under gentle orbital shaking with secondary antibody: Goat anti-mouse IgG AlexaFluor 488 Superclonal Recombinant Secondary antibody (ThermoFisher Scientific #A28175) at 1:2000 dilution. Slides were washed five times for 10 min with PBST, then washed three more times with PBS before mounting overnight with 4',6-diamidino-2-phenylindole (DAPI). All antibodies were incubated with 5% BSA in 0.1% Tween-PBS. Immunofluorescence images were taken at 63x objective with a Zeiss LSM 780 confocal microscope in 5–10 slices, with maximum intensity projections across the entire image plane generated in Zeiss ZEN 2010 for figures.

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.