GSM8224185: CM26 Unselected A; Homo sapiens; OTHER
Genomic DNA was isolated using the QIAamp DNA Blood Mini Kit (Cat #51185, Qiagen), and gRNA sequences were amplified and prepared for Illumina sequencing via the NextSeq (genome-wide screen) or MiSeq (targeted screen) platforms. Genomic DNA was amplified by a two-step PCR to amplify sgRNA sequences (genome-wide screen PCR1: 18 cycles PCR 2: 20 cycles, targeted sub-library screen PCR1: 12 cycles PCR2: 20 cycles). One thousand- or five hundred-fold coverage (for the genome-wide and targeted sub-library screens, respectively) of the sgRNAs were amplified with maximum of 2 ug of genomic DNA per reaction.  PCR1 reactions of each sample were pooled and either used directly for PCR2 (genome-wide screen) or first cleaned up with a QIAquick PCR Purification Kit (targeted sub-library screen) before PCR2, which include Illumina barcodes and adaptor sequences. PCR2 products were then pooled, run via gel electrophoresis, and bands were extracted and cleaned up using the QIAquick Gel Extraction Kit. Targeted sub-library samples were then purified by double-sided bead cleanup (AMPureXP, Beckman Coulter). Samples were quantified with a Qubit dsDNA HS Assay and pooled.