Quickextract dna extraction solution

Genomic DNA was extracted from both cells and tissue using Quick Extract DNA extraction solution (Epicenter) following the recommended protocol. Treated cells and tissues were used as a template for PCR for both captured sequencing and SURVEYOR nuclease assay using high-fidelity polymerases (Thermo Scientific) as previously described (Hsu et al., 2013 (link)). Genomic PCR products were subjected to library preparation using the Nextera XT DNA Sample Prep Kit (Illumina) or using customized barcoding methods. Briefly, low-cycle, first-round PCR was performed to amplify the target site. Second-round PCR was performed to add generic adapters, which were then used for a third round of PCR for sample barcoding. Samples were pooled in equal amounts and purified using QiaQuick PCR Cleanup (QIAGEN), quantified using Qubit (Life Technologies). Mixed barcoded library was sequenced on an Illumina MiSeq System.

To start, 2×10⁵ U2OS.eGFP.PEST cells were nucleofected with either 20 pmol of SpCas9 or 20 pmol of preformed SpCas9:gRNA ribonucleoprotein complex. Approximately, 1×10⁶ cells per well in a 12-well format were plated in the absence or presence of the compound at the indicated concentrations in two biological replicates. Cells were harvested at 10, 12, 14, and 18 h, and genomic DNA was extracted using the QuickExtract™ DNA extraction solution (Epicentre) by incubating the cells at 65°C for 15 min, 68°C for 15 min, and 98°C for 10 min. Next-generati on sequencing samples were prepared in a two-step PCR (Supplementary Table S1) following a reported protocol. In the first step, the PCR was performed using primers that amplified the target eGFP genomic loci of interest and also introduced an adapter priming sequence for the second-step PCR. The second-step PCR attached Illumina P5 adapters with barcodes, after which the PCR products were isolated via a two-step gel-purification protocol. DNA concentrations were determined using the Qubit® dsDNA HS Assay Kit (Life Technologies), and sequencing of the pooled samples was performed using a single-end read from 280 bases using the MiSeq Reagent Kit v2 300 (Illumina) according to the manufacturer’s protocol. The percentage of indel frequencies was calculated by analyzing the demultiplexed sequence files using MATLAB.