GUIDE-seq Library Preparation Protocol

GUIDE-seq library preparation was performed as previously described^{12 (link)}. Briefly, genomic DNA was purified with Agencourt DNAdvance using a BioMek Fx^P automation system (both from Beckman Coulter). Genomic DNA was sheared to average fragment size of 500 bp by Covaris E220 sonication (Covaris) and purified with SPRI magnetic beads. Genomic DNA was quantified by Qubit (Invitrogen), and 400 ng was used for GUIDE-seq library preparation. Genomic DNA was treated with End-repair Mix (Qiagen) and A-tailed with Taq polymerase (Fisher), ligated to single-tailed sequencing adapters and purified with SPRI magnetic beads. The adapter-ligated library was subjected to two rounds of nested PCR using dsODN sense- and antisense-specific primers in separate reactions for each sample, and then purified with SPRI magnetic beads. Libraries were quantified with Kapa qPCR Library Quantification kit (Kapa). Equimolar amounts of samples were pooled for GUIDE-seq libraries were sequenced with 150 bp paired end reads on Illumina NextSeq 550 or HiSeq 2500 sequencers. GUIDE-seq analysis was performed as previous described^{12 (link)}.