GSM7928036: CAST-seq on non-activated T cells treated with Cas9 targeting CD4 and PD1 donor2; Homo sapiens; OTHER
Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Cat. No./ID:  69504) according to the manufacturer's protocol. 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 700 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, 63°C (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.