DNA STAT-60 Kit Genomic DNA was extracted from each sample (detailed extraction methods are described in supplemental methods). The sgRNA regions were then amplified and indexed for Illumina sequencing using a one-step PCR method and primers specific to the LentiCRISPRv2-based Brunello libraries. Primers and indices used for the generation of amplicon libraries are listed in Table S1. Brunello  DNA samples were amplified in ten 100 µl reactions using the NEBNext High-Fidelity 2X Master Mix Kit (New England Biolabs), 0.5 µM of forward and reverse primers and 10 µg of DNA template per reaction with the following program: initial denaturation at 98°C for 3 min, 24 cycles of denaturation at 98°C for 10 s, annealing 60°C for 15 s and extension 72°C for 25 s, and final extension at 72°C for 2 min. 256 bp amplicons were quantified on a 2% agarose gel stained with SYBR Safe (Invitrogen), using the Gel Doc quantification software (Bio-Rad). Amplicons were first pooled in an equimolar fashion and then the pools were gel-extracted using the PureLink Quick Gel Extraction Kit (Thermo Fisher Scientific). The sequencing was carried out at the Interdisciplinary Center for Biotechnology Research (ICBR; University of Florida) using a NovaSeq 6000 sequencer (Illumina). The Brunello amplicons were sequenced using the S4 2X150 cycles Kit (Illumina)