title: GSM9695655: LDLR-FL library, FACS sorted top 20% GFP-positive cells, reporter readout, read length 79+43, rep 2; Homo sapiens; OTHER
library_name: GSM9695655
library_construction_protocol: After FACS, cell gDNA was extracted via the PureLink Genomic DNA kit (Invitrogen). gDNA was subject to PCR to amplify the region spanning the pegRNA and activity reporter or the endogenous locus (LDLR137-219), or only the pegRNA and the activity reporter (LDLR-FL). After this initial PCR reaction (PCR1), samples with similar CTs as determined by qPCR were pooled, and DNA was subject to another round of PCR (PCR2), with the number of cycles being 2-3 cycles less than the qPCR CT to add i5 sequencing adapters to enable next-generation sequencing. After PCR2, purified pools are run on a Tapestation (Agilent) using a D1000 ScreenTape, and moles in each sample are used to pool all samples together, after which the final pool is purified using SPRIselect beads.
identifiers.namespace: GEO
identifiers.id: GSM9695655_r1