title: GSM8319610: GIN440.TSC22D1/TSC22D2.T2B; Homo sapiens; OTHER
library_construction_protocol: gRNA abundance was measured at T0 and endpoints of each screen by next-generation sequencing (NGS). Genomic DNA was extracted from cell pellets using the Wizard Genomic DNA Purification kit (Promega). Sequencing libraries were prepared from 50 µg gDNA (200-fold library coverage of haploid genome) via a 2-step nested PCR using primers that include Illumina TruSeq adapters with i5 and i7 indices. Barcoded libraries were gel purified using PureLink Quick Gel Extraction kit (Thermo Fisher Scientific) and sequenced on an Illumina HiSeq2500 or NovaSeq6000 using single-read sequencing and standard primers for dual-indexing with HiSeq SBS Kit v4 or NovaSeq v1.5 reagents. T0 samples were sequenced at read depths of 500-fold library coverage, while all other samples were sequenced at 200-fold. Amplicons were sequenced using a custom sequencing recipe comprising DC: 21 cycles; R1: 36 cycles; Index read 1: 8 cycles; Index read 2: 8 cycles.