title: GSM5230448: Amplicon_Plate_2_Donor_1_miniseq; Homo sapiens; OTHER
library_construction_protocol: On day 5 (sgRNA #1 Donor 1-3) or day 7 (sgRNA #2 Donor 1, 3) post-electroporation, genomic DNA was isolated from each sample using DNA QuickExtract (Lucigen, Cat #QE09050) according to the manufacturer's protocol. Primers were designed to flank each sgRNA genomic target site. Amplicons containing CRISPR edit sites were generated by adding 1.25 µL each of forward and reverse primer at 10nM to 5 µL of sample in QuickExtract, 12.5 µL of NEBNext Ultra II Q5 master mix (NEB, Cat #M0544L), and H2O to a total 25 µL reaction volume. The following PCR cycling conditions were used: 98°C for 3 minutes, 15 cycles of 94°C for 20 seconds followed by 65°C-57.5°C for 20 seconds (0.5°C incremental decreases per cycle), and 72°C for 1 minute, and a subsequent 20 cycles at 94°C for 20 seconds, 58°C for 20 seconds and 72°C for 1 minute, and a final 10 minute extension at 72°C. Samples were then diluted 1:200 and subsequently Illumina sequencing adapters and indices were added in a second PCR reaction. Indexing reactions included 1 µL of the diluted PCR1 sample, 2.5 µL of each the forward and reverse indexing primers at 10 µM each, 12.5 µL of NEB Q5 master mix, and H2O to a total 25 µL reaction volume. The following PCR cycling conditions were used: 98°C for 30 seconds, followed by 98°C for 10 seconds, 60°C for 30 seconds, and 72°C for 30 seconds for 12 cycles, and a final extension period at 72°C for 2 minutes. Samples were quantified in a 96-well plate reader using the Quant-IT DNA high sensitivity assay kit (Invitrogen, Cat #Q33232) according to the manufacturer's protocol and pooled into 1 tube. Post pooling, samples were then SPRI purified, and quantified using an Agilent 4200 TapeStation. Samples were then sequenced on an Illumina MiniSeq or NextSeq with PE 300 reads.