oligo sequencing of gRNA library
The 1062 PAM library oligo was synthesized using array synthesis and cloned as a pool into the transfer plasmid for lentivirus production as previously described. Viral vectors were produced in HEK293T cells and assayed viral titer by flow cytometry. HEK293T cells stably expressing SpCas9 were infected at a MOI=0.3 to ensure that most cells receive only one viral construct with high probability. At day 1, transduced cells in each dish were split into 2 dishes equally. At day 2, the sub-group 1 was changed to fresh medium containing 50 g/ml hygromycin + 1 g/mL puromycin. The sub-group 2, instead, was changed to medium containing 50 g/ml hygromycin + 1 g/mL puromycin + 1 g/mL doxycycline. After 6 and 10 days of selection, cells were harvested to extract genomic DNA, which was subjected to PCR for amplification of the gRNA-target-DNA surrogates. The PCR products were purified by 1.5% gel and pooled in equal amounts. Deep amplicon sequencing was conducted and raw data was pre-processed and assembled as previously described. Further data pre-processing is detailed below.