Dneasy tissue kit

Genomic DNA was purified from the retina of rd12 mice with a DNeasy Tissue kit (Qiagen) according to the manufacturer’s instructions. Genomic DNA (8 μg) was incubated with purified Cas9 protein (100 nM) and sgRNA (300 nM) in a reaction volume of 400 μl for 8 hours at 37°C in a buffer [100 mM NaCl, 40 mM tris-HCl, 10 mM MgCl₂, and bovine serum albumin (100 μg/ml; pH 7.9)]. Digested genomic DNA was purified again with a DNeasy Tissue kit (Qiagen) after RNase A (50 μg/ml) was added to remove sgRNA. To check targeted in vitro DNA cleavage, digested genomic DNA was mixed with 2× SYBR Green Master Mix and analyzed by real-time quantitative PCR (qPCR) using forward primer 5′-GGACATTCTACATATGAATCCAGG-3′ and reverse primer 5′-TAACATTCTCAGGTGGCTGTGCAA-3′. The fraction of target sites that were cleaved was measured using the comparative C_T method (33 (link)).
For Digenome-seq, genomic DNA (1 μg) was sonicated to produce fragments in the 400- to 500-bp range using the Covaris system (Life Technologies) and blunt ended using End Repair Mix (Thermo Fisher). Fragmented DNA was ligated with adapters to produce libraries, which were then subjected to whole-genome sequencing (WGS) using a HiSeq X Ten Sequencer (Illumina) at Macrogen. WGS was performed at a sequencing depth of 30× to 40×, and the DNA cleavage score was calculated using a previously published scoring system (34 (link)).

The genomic DNA was extracted using the DNeasy Tissue kit (Qiagen) following the manufacturer’s protocol. SpCas9 nuclease, at a concentration of 100 mM, was combined with AB_T1, BC_T2, and BC_T3 sgRNAs, each at a concentration of 100 nM, and the mixture was incubated at room temperature for 30 min. This mixture was then added to 10 μg of genomic DNA in a 1000ul reaction volume containing 100 mM NaCl, 50 mM MgCl₂, and 100 μg/mL BSA. After incubating the reaction mixture at 37 °C for 8 h, the digested genomic DNA underwent a secondary purification step using the DNeasy Tissue kit (Qiagen). During this purification process, RNase A (50 μg/mL) was added to remove any residual sgRNA. Cas9-mediated digested genomic DNA was subjected to WGS with a sequencing depth of 30×–40× using an Illumina HiSeq X Ten Sequencer at Macrogen. The genome sequence was mapped using the Isaac aligner and DNA cleavage sites were identified using the Digenome program, which is available at https://github.com/chizksh/digenome-toolkit2.