GSM2545411: HER2 KRAB A431 day 19 Low Rep 3; Homo sapiens; OTHER
An aliquot of 1.3x10^6 cells was taken before sorting for a bulk unsorted sample for each screen. The highest and lowest 10% of cells were sorted based on mCherry signal for the dCas9KRAB screen of HBE1 in K562 cells, or Alexa Fluor 488 signal for all HER2 screens. 1.3x10^6 cells were sorted for each group. Cell sorting was performed using a MoFlo Astrios (Beckman Coulter) or SH800 (Sony Biotechnology). After sorting, cells were harvested for genomic DNA using the DNeasy Blood and Tissue Kit (Qiagen). To amplify the gRNA libraries from each sample, 8.3 µg of gDNA was used as template across 8 100 µL PCR reactions using Q5 hot start polymerase (NEB). Amplification was carried out following the manufacturer’s instructions using 25 cycles at an annealing temperature of 60C. Amplified libraries were purified using Agencourt AMPure XP beads (Beckman Coulter) using double size selection of 0.65X and then to 1X the original volume. Each sample was quantified after purification using the Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher).