GSM7124235: 175_S1; Homo sapiens; OTHER
Genomic DNA from HEK293T cells was isolated using a Gentra Puregene Kit (QIAGEN) according to the manufacturer's instructions. Purified genomic DNA was sheared with a Covaris S2 instrument to an average length of 300 bp. The fragmented DNA was end repaired, A-tailed and ligated to an uracil-containing stem-loop adaptor, using the KAPA HTP Library Preparation Kit, PCR Free (KAPA Biosystems). Adaptor-ligated DNA was treated with Lambda Exonuclease (NEB) and E. coli Exonuclease I (NEB) and then with USER enzyme (NEB) and T4 polynucleotide kinase (NEB). Intramolecular circularization of the DNA was performed with T4 DNA ligase (NEB) and residual linear DNA was degraded by Plasmid-Safe ATP-dependent DNase (Lucigen). In vitro cleavage reactions were performed with 125 ng Plasmid-Safe-treated circularized DNA, 90 nM Cas9-NG protein, Cas9 nuclease buffer (NEB) and 90 nM synthetic chemically modified sgRNA (-175), in a 50 μl volume. Cleaved products were A-tailed, ligated with a hairpin adaptor (NEB), treated with USER enzyme (NEB) and amplified by PCR with barcoded universal primers (NEBNext Multiplex Oligos for Illumina (NEB), using Kapa HiFi Polymerase (KAPA Biosystems).