GSM7660732: TF-1, CRIPSRgate screen, 14 days, rep3; Homo sapiens; OTHER
After 14 days post induction of ZIM3-Cas9 expression cells were harvested in a 1000x library representation and genomic DNA for the three single cell clones was isolated with phenol-extraction using PhaseLock tubes, followed by ethanol precipitation. Multiple parallel 50 µL PCR reactions, each containing 1 µL gDNA template adding up to 7 µg gDNA, were performed to maintain sgRNA representation. sgRNAs were PCR amplified using the AmpliTaq Gold Polymerase (Life technologies). In a first round of PCRs, random barcodes and sample barcodes were added to the sgRNA sequences. PCR products for each single cell clone were combined and purified using the NucleoSpin Gel and PCR clean-up kit (macherey-Nagel). Afterwards using a second round PCR, using similar cycling conditions with the exception of 10 ng input as template and 7 total cycles the standard Illumina P7 and P5 adaptors were added. The final libraries were cleaned up from a 2% agarose gel, pooled and analyzed on an P2 flow cell (400 mio reads).