title: GSM7635443: SEMCas9-Day7-Rep1-SFM004-2583705; Homo sapiens; OTHER
library_construction_protocol: A set of ~5700 sgRNA oligos that target 229 HOXA9 binding peaks were designed for array-based oligonucleotide synthesis (CustomArray). The unique binding of each sgRNA was verified by sequence blast against the whole human genome in the sgRNA pooled library. The synthesized oligo pool was amplified by PCR and cloned into the LentiGuide-Puro-IRES-CFP backbone by the in-fusion assembly (Clontech #638909). The Cas9-expressing MLL-r SEM cell line was infected by pooled sgRNA library at low M.O.I (~0.3). Infected cells were selected by blasticidin and puromycin. After cell recovery from selection, the pooled population was collected on day 0 and maintained in liquid culture for 7, 14, and 21 days. The sgRNA sequences were recovered by genomic PCR analysis and deep sequencing using Novaseq for paired-end 150 bp read length (Illumina). The primer sequences used for cloning and sequencing are listed in Supplementary file 1. The sgRNA sequences are described in Supplementary file 2. High-titer lentivirus stocks were generated in 293 T cells, as previously described (Vo et al., 2017). CRISPR