title: GSM7795451: MigLib-D21; Homo sapiens; OTHER
library_construction_protocol: Following these two rounds of migration we trypsinized and collected the cells that either migrated twice or did not migrate twice (with a similar number of cells, 24%, being found to have migrated during this second round) and isolated gDNA using DNeasy kits (Qiagen). All gDNA was split between sample-indexed 100uL Q5 PCR reactions (up to ~340ng max input per 100uL reaction) to amplify out the gRNA protospacer from HFF cells. These PCRs from gDNA were run as follows [ 98C for 30s / 25x: 98C for 10s, 60C for 30s, 72C for 15s / 72C for 2 min] with primers in Supplementary Table 15, followed by individual PCRs being pooled together and subjected to a double-sided 0.65X/1X SPRI clean-up. Quality control was performed by running the amplified libraries out on a High Sensitivity D1000 Tapestation (Agilent) to confirm expected size, and Qubit dsDNA HS assays were performed to determine a final concentration. All libraries were pooled to an effective concentration of 4 nM and combined in equal volumes prior to sequencing on an Illumina MiSeq, using a v2 50 cycle reagent kit with Read1 being 21 cycles (protospacer) and index read 1 being 6 reads (sample barcoding).