GSM7813791: Optimization;RPE1;opCas12a;T21C; Homo sapiens; OTHER
Genomic DNA was extracted from the cell pellets collected at the start (T0) and end time points using the Wizard Genomic DNA Purification Kit (Promega #A1120). First, the lentiviral integrated hgRNA expression cassettes were amplified from genomic (g)DNA equivalent of ~ 100-fold library coverage using NEBNext Ultra II Q5 Master Mix (New England Biolabs #M0544X). Individual 50 µL PCR reactions containing 3.5 μg gDNA were performed using PCR1_Hybrid_Outer_F2 and PCR1_Hybrid_Outer_R1 primers for the optimization and exon deletion screens and primers A265 and PCR1_BE_Rev for the base editor screens, respectively (see Table S3). After pooling the individual PCR-1 reactions, a fraction was purified using a PCR purification column and used as template for PCR-2, in which each sample was barcoded with unique i5 and i7 index primer combinations. The resulting PCR-2  products were resolved on a 2% agarose gel using SYBR Safe DNA stain (ThermoFisher Scientific #S33102). The desired band was excised and subjected to gel extraction (ThermoFisher Scientific #K0691). Quantification of the extracted libraries was conducted using the Qubit dsDNA HS assay (ThermoFisher Scientific #Q32851) and Tape Station (Agilent #5067-5582 and #5067-5583). The quantified and validated sequencing libraries were pooled, and paired-end sequencing was performed on either an Illumina NextSeq 500 or a NovaSeq 6000 platform.