title: GSM9343569: Trial1_PreTreatment_Ctrl; Homo sapiens; OTHER
library_construction_protocol: gDNA was extracted from cells using the Quick-DNA Miniprep Kit (Zymo) Libraries were constructed for sequencing the CRISPR sgRNA region using a two-step pcr process.  Genomic DNA (100 to 5000 ng) was PCR amplified for 14 cycles using custom CRISPR PCR primers with NEBNext Ultra II Q5 Master Mix (NEB, M0544) (Forward Primer: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTGTGGAAAGGACGA AACACCG; Reverse Primer: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCAATTCCCACTCC TTTCAAGACCT). This was performed as 8 replicate reactions with the amplicons being pooled before the next step. Dual unique indexing adapters (Illumina, 20027213) (Read 1 Adapter: CTGTCTCTTATACACATCT; Read 2 Adapter: CTGTCTCTTATACACATCT) were added to the CRISPR amplicons during a second PCR with 6 cycles and performed as 3 replicate reactions. Libraries were pooled and sequenced on an Illumina NovaSeq X instrument as 150 x 150 cycle paired-end sequence runs.