title: GSM7876498: Untreated sample used as control donor 3 replicate 1; Homo sapiens; OTHER
library_name: GSM7876498
library_construction_protocol: Between 2 and 4 days post targeting, gene edited cells were harvested and QuickExtract DNA extraction solution (Epicentre) was used to collect gDNA. CAST-Seq was performed as previously described (Turchiano et al, 2021) with minor modifications. Briefly, genomic DNA was fragmented by enzymatic digestion (NEBNext® Ultra™ II FS DNA Library Prep Cat.# E7805S) to obtain average fragment lengths of 350-500 bp. After linker ligation (NEBNext®Ultra™ II FS DNA Library Prep Cat.# E7805S) and DNA purification, two rounds of PCR utilizing Q5 polymerase (NEB, Cat.# M0493S) were performed with the following conditions: 20 cycles at 95°C for 15 s, 61°/63°C (g1 or g1+2/g2, first reaction) or 68°C (second reaction) for 20 seconds, 72°C for 20 seconds. A third PCR introduced the barcoded Illumina adapter for sequencing (NEB, NEBNext Multiplex Oligos for Illumina, Cat.# E7335, E7500, E7710, E7730). Libraries were sequenced by Azenta Life Sciences using the Illumina NovaSeq platform with a 2 x 150 bp configuration.