GSM5702976: CD37_enhancer_amplicon_CD37_high_Donor1; Homo sapiens; OTHER
Five days after electroporation, cells were stained for CD37 (Miltenyi Biotec Cat# 130-123-268) 1:50. The cells were sorted into the 15% lowest and 15% highest expressing cells. Genomic DNA was isolated from each sample using DNA QuickExtract (Lucigen, Cat #QE09050) according to the manufacturer's protocol. Amplicons around the SNP were generated by adding 2.5 µL each of forward (cgacgctcttccgatctCAGAGGCCACTGGGTGTG) and reverse primer (cgtgtgctcttccgatctCAGCCTGTTCTGGACCAGG) at 10 uM to 20 µL of sample, 25 µL of NEB Ultra II Q5 master mix (NEB, Cat #M0544L). The following PCR cycling conditions were used: 98°C for 30 seconds, followed by 98°C for 10 seconds, 67°C for 30 seconds, and 72°C for 30 seconds for 35 cycles, and a final extension period at 72°C for 2 minutes. 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 the forward and reverse indexing primers 10 µM each, 12.5 µL of NEB Ultra II 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, 65°C for 1:15 for 15 cycles, and a final extension period at 65°C for 5 minutes. Samples were cleaned up with a 1X SPRI reaction and sequenced on a NovaSeq with paired end 150bp reads. Amplicon sequencing