GSM5155215: ABE_HBF_LOW_rep1; Homo sapiens; OTHER
Genomic DNA was extracted with the PureLink Genomic DNA Mini Kit (Life Technologies). sgRNAs were amplified for 27 cycles, using barcoded lentiGuide-Puro–specific primers with 400 ng of genomic DNA per reaction. The PCR products were then pooled for each sample. The barcoded libraries were pooled at an equimolar ratio, gel purified, and subjected to massively parallel sequencing with a MiSeq instrument (Illumina) Oligonucleotide PCR products were gel purified and cloned into a BsmBI-digested and gel-purified pLentiGuide-Puro5 vector (Addgene cat. #52963), which encodes a puromycin resistance cassette, by Gibson assembly using NEBuilder HiFi DNA Assembly Master Mix (NEB). The assembly products were transformed into NEB stable competent cells, then the plasmids were extracted. Lentiviral particles were generated by transfecting HEK293T cells with the library plasmid, pVSVG, pRevtat, and pGagpol, using Lipofectamine 2000 (Life Technologies). Viral vector titers were determined by serial dilution.