title: GSM9653960: HGC27, no NK, rep4; Homo sapiens; OTHER
library_name: GSM9653960
library_construction_protocol: Surviving cancer cells from both NK and no NK conditions were collected and sorted by FACS for GFP+/mcherry+ cells. The cells were pelleted at 300 x g for 5 min, the supernatant removed, and stored in -80C for further processing.  Genomic DNA was isolated using Quick-DNA Midiprep Plus Kit (Zymo Research #D4075). gRNA sequences were amplified from genomic DNA using the NEBNExt High-Fidelity 2X PCR Master Mix (NEB #M0541) with 50 uL reaction volumes and 2.5 ug of DNA per reaction and mixed staggered primers (Forward Primer: 5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT[Stagger, 0-7nt]TTGTGGAAAGGACGAAACACC-3' Reverse Primer: 5'-CAAGCAGAAGACGGCATACGAGAT [8nt-barcode]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCTACTATTCTTTCCCCTGCACTGT-3'). PCR reactions were pooled and column purified using Monarch PCR and DNA Cleanup Kit (NEB #T1030S). A fraction of the eluate was run on a gel, the band corresponding to the library amplicon excised, and then column purified. Amplified products were quantified using the Qubit 1X dsDNA HS Assay (Thermo Scientific #Q33230), normalized by concentration and sequenced on an Illumina NextSeq using 500/550 v2.5 kits at 1000X coverage per gRNA.
identifiers.namespace: GEO
identifiers.id: GSM9653960_r1