HEK 293FT cells plated in 96-well plates were transfected with Cas9 plasmid DNA and sgRNA PCR cassette 72 h before genomic DNA extraction (Supplementary Fig. 4). The genomic region flanking the CRISPR target site for each gene was amplified (Supplementary Fig. 6, Supplementary Table 5 and Supplementary Sequences) by a fusion PCR method to attach the Illumina P5 adapters as well as unique sample-specific barcodes to the target amplicons (schematic described in Supplementary Fig. 5). PCR products were purified using EconoSpin 96-well Filter Plates (Epoch Life Sciences) following the manufacturer's recommended protocol.
Barcoded and purified DNA samples were quantified by Quant-iT PicoGreen dsDNA Assay Kit or Qubit 2.0 Fluorometer (Life Technologies) and pooled in an equimolar ratio. Sequencing libraries were then sequenced with the Illumina MiSeq Personal Sequencer (Life Technologies).