title: SMRT-seq of HUDEP2-GFP/BFP cell model
design: The first PCR reaction (PCR1) was used to target 6-kb region around the Cas9 cut site and tag template molecule with adapter using a tailed primer pair. The PCR1 reaction contained 100 ng of gDNA, 200 nM of each tailed primer in 50 l of reaction (LongAmp Hot Start Taq 2 Master Mix, NEB). The PCR1 program consisted of initial denaturation (2 min at 94C) and two cycles of denaturation (30 s at 94C), annealing (30 s at 60C), and extension (6 min at 65C). After completion of PCR1. The PCR1 product was purified using SPRIselect (Beckman Coulter, B23317) and eluted in 30 l of water. In second PCR reaction (PCR2), barcodes are incorporated by using universal sequences tailed with 16-bp PacBio barcode sequences (Sequal_RSII_96_barcodes_v1). The PCR3 reaction contained X ng of PCR1 product, 200 nM of each barcoded universal primer in 100 l of reaction (LongAmp Hot Start Taq 2 Master Mix). The PCR1 program consisted of initial denaturation (2 min at 94C) and 5 to 10 cycles of denaturation (15 s at 94C), annealing (30 s at 60C), and extension (6 min at 65C) followed by the final extension (5 min at 65C). The minimum cycle number (10 cycles) was used to obtain sufficient PCR product (>100 ng) for library preparation. The PCR2 product was purified using SPRIselect and eluted in 30 l of water. One hundred nanograms of barcoded amplicon from PCR2 was pooled, and a total of x g of the pooled amplicons was used for PacBio library preparation, which consists of DNA damage repair, end repair/A-tail, SMRTbell adaptor ligation (SMRTbell Express Template Prep Kit 2.0), nuclease treatment (SMRTbell Enzyme Clean Up Kit), and AMPure bead purification following the standard protocol. The SMRTbell library was sequenced on a PacBio Sequel II 8M flow cell in CCS mode following the standard protocol with 1 hour of preextension and 30 hours of collection time (PacBio). The PacBio subreads were converted to HiFi reads, and Q20 CCS reads were used for analysis. GFP and BFP allele specific editing rate was quantified by a custom python script.