GSM7849070: CAST-seq on samples treated with SaCas9 paired with g1 replicate2; Mus musculus; OTHER
Cells were harvested on day 6 post nucleofection for genomic DNA extraction using the NucleoSpin Tissue Mini kit for DNA from cells and tissue (Macherey-Nagel REF 740952.250). 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.