GSM3928095: U937 infected with Y. pestis WT and lcrV mutant, targeting mutant; Homo sapiens; OTHER
Genomic DNA was extracted with a Blood & Cell Culture Midi kit (Qiagen) and used to prepare a sgRNA library with a two-step PCR protocol. The first PCR was used to amplify the sgRNA containing cassette. Amplified products were subjected to a second PCR with a primer pair encoding a unique 8-bp barcode required for multiplexing along with a stagger sequence to increase library complexity. The resulting amplicons from the second PCR were gel extracted, quantified, mixed and sequenced using a HiSeq 2500 (Illumina). The pooled human GeCKO v2.0 lentiviral library was obtained from Addgene (Feng Zhang, Massachusetts Institute of Technology, Cambridge, MA). The GeCKO v2.0 lentiviral library expresses RNA guided endonuclease Cas9 and short guide RNAs (sgRNA) targeting 19,052 genes (three sgRNAs per gene).
GEO
GSM3928095