title: NGS of homo sapiens: SHSY5Y GECKO
design: PCR on the gDNA samples to amplify the sgRNA guide sequence region and to append the Illumina (HiSeq) compatible adapters and barcodes. A two-step PCR was performed: First, the amount of input gDNA for each sample was calculated in order to achieve 500X library coverage. For each replicate sample, 22 separate 100 l PCR reactions with 10 g of input genomic DNA in each reaction with Herculase II Fusion DNA Polymerase (Agilent, Cat#600677) were completed in an ABI Veriti Thermal Cycler (Applied Biosystems). Amplicons from the first PCRs were then pooled together and mixed thoroughly to be used as the template for the second PCR reaction. The second PCR was designed to attach standard Illumina adaptors and unique barcodes to the amplicons from the first PCR to allow for HiSeq reactions and demultiplexing of the reads, respectively. The second PCR was conducted on 5 l of the first PCR amplicon as the template, in 100 l reactions with a total of 22 reactions. Primers for the second PCR (Table S2) were synthesized as IDT DNA Ultramer Oligonucleotides (Integrated DNA Technologies) and also included a staggered region with variable lengths of sequence to enhance the library complexity.