GSM3770848: CRISPR-screen.tiling.CRKO_T14_Rep2; Homo sapiens; OTHER
Oligonucleotide libraries were synthesized by Agilent and then cloned into an sgRNA expression vector (pMCB320) that had been cut with BstXI and BlpI restriction enzymes, by ligation, using the T4 Ligase. Large scale lentivirus production and infection of K562-Cas9 cells were performed as previously described (PMID: 23394947 and PMID: 23739767). Selection with puromycin was started three days after infection and continued for 3--4 days until the mCherry-positive  percentage of cells was greater than 80%, as observed by flow cytometry on a BD Accuri. Cells were maintained in log growth conditions each day by diluting cell concentrations back to a 0.5 x 10^6 cells/mL. Genomic DNA was extracted following Qiagen's Blood Maxi Kit, and the guide composition was sequenced and compared to the plasmid library using casTLE version 1.0 (available at https://bitbucket.org/dmorgens/castle.)