Genomic DNA Isolation and sgRNA Library Preparation

Genomic DNA was isolated using QiaAmp DNA Blood Maxi or QiaAmp DNA mini kits (Qiagen) according to the manufacturer’s instructions, genomic DNA was then amplified using Herculase II polymerase (Agilent) as described previously (Deans et al., 2016 (link)). To prepare the sgRNA sequencing library, the integrated sgRNA-encoding constructs were PCR amplified using Agilent Herculase II Fusion DNA Polymerase, followed by a second PCR amplification introducing sample-specific Illumina index barcodes and adapters for deep sequencing. Deep sequencing was performed using the MiSeq Reagent Kit v2 (300 cycles; Illumina) employing a custom sequencing oligo according to the manufacturer’s instructions:
(5’-GCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTAAACTTGCTATGCTGTTTCCAGCTTAGCTCTTAAAC-3’).