title: GUIDE-seq of SpCas9-expressing HEK293 with modified TRAC-G-sgRNA read 1
design: GUIDE-Seq for unbiased identification of CRISPR-Cas off-targets was performed as previously described (Tsai SQ et al, Nat Biotechnol 2015) . Briefly, SpCas9-expressing HEK293 cells were co-transfected with dsDNA oligonucleotides (GUIDE-seq tag, for sequence Tsai SQ et al 2015) and the respective sgRNA. Subsequently, genomic DNA was isolated after 72 hours using QuickExtract (Lucigen, USA) and fragmented using the LOTUS DNA library kit (IDT, Coralville, USA). Libraries were generated according to the original protocol (Tsai SQ et al 2015), followed by Illumina based next generation sequencing. Read alignment and data analysis was performed based on GUIDE-seq software (Tsai SQ et al 2015).