Hap1 cells

Human, CML-derived HAP1 cells (Horizon Discovery) were used for generation of a PHD1/PHD3 double knockout cell line. Cells were grown in Iscove's DMEM supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were cotransfected with a combination of pHL-EF1a-SphcCas9-iP-A, pMLM3636-PHD1gRNA2, and pMLM3636-PHD3gRNA2. Twelve hours after transfection, cells were selected using 10 μg/ml puromycin. Following 48 hours of selection, cells were serial diluted to isolate clonal populations which were then screened for mutation of PHD1 and PHD3.
Initial clone screening was done by Surveyor Mutation Detection Kit (Integrated DNA Technologies) following the manufacturer's protocol. PCR primers used for heteroduplex production from the PHD1 gene were PHD1 ex3 screen 5′: 5′-GGT TTG GGG TAT CTT GAA ACA A-3′, and PHD1 ex3 screen 3′: 5′-GGG AAA GTG TTC ATT ACA AGG C-3′. For the PHD3 gene, the PCR primers were PHD3 ex2 screen 5′: 5′-GGC ATA CAA ACA GGT TAT GCA A-3′, and PHD3 ex2 screen 3′: 5′-TTA CCT TCC CTT CAT CAA CCA C-3′. Following mutation detection, Sanger sequencing was performed on these PCR products to confirm mutation. Real-Time PCR, described later, was used to confirm loss of transcript abundance relative to HAP1 control cells.