title: GUIDE-seq_TRAC2: HEK293T cells
design: 4e5 HEK293T cells were transfected with 2 g pX330 plasmids and 5 pmol dsODN using Lonza 4D-Nucleofector. Cells were harvested three days post-transfection. Genomic DNA was extracted using HiPure Tissue DNA Kit (Magen) and fragmented to an average size of 200bp by Covaris S200 instrument. Fragmented DNA was purified using VAHTS DNA Clean Beads (Vazyme). Library was prepared through end repair, A-tail addition, and adapter ligation using VAHTS Universal DNA Library Prep Kit (Vazyme). The adapter-ligated DNA was subjected to two rounds of nested PCR using dsODN sense- and antisense- specific primers in separate reactions. Libraries were sequenced with paired-end 150 on Illumina platform.