Herculase 2

ESCs were lysed in genomic lysis buffer (10 mM Tris, pH 7.5, 10 mM EDTA, 0.5% SDS, and 400 μg/ml proteinase K) for at least 2 h at 55 °C. After proteinase K heat inactivation at 95 °C for 15 min, 0.5 volume of 5 M NaCl was added, and samples were centrifuged for 10 min at 15,000 r.p.m. Supernatants were mixed with one volume of isopropanol, and DNA precipitates were washed in 70% EtOH before resuspension in 10 mM Tris, pH 8.0. Cas9-induced mutations were detected using the T7 endonuclease I. Briefly, the target region surrounding the expected mutation site was PCR amplified using Herculase II (600675, Agilent Technologies). PCR products were column-purified (Qiagen) and subjected to a series of melt–anneal temperature cycles with annealing temperatures gradually lowered in each successive cycle. T7 endonuclease I was then added to selectively digest heteroduplex DNA. Digest products were visualized on a 2.5% agarose gel.

Screening-scale infections were performed with virus in the 12-well format and infected wells were pooled 24 hr post-centrifugation. Infections were performed with 3 replicates per treatment arm, and a representation of at least 1000 cells per SHOC2 variant was achieved following puromycin selection. Approximately 3 days after infection and selection, all cells within a replicate were pooled and split into Falcon™ Cell Culture Multi Flasks flasks and treated in media with 10 nM trametinib or DMSO control. Cells were passaged in fresh media containing drugs or vehnicle control (DMSO) every 3-4 days. Cells were harvested 16 days after initiation of treatment and gDNA extracted (Genomic DNA Extraction Kit, Machery-Nagel). Twelve PCR reactions were performed for each gDNA sample. The volume of each PCR reaction was 100 μl and contained ~3 μg of gDNA. Herculase II (Agilent) was used as the DNA polymerase. All 12 PCR reactions for each gDNA sample were pooled, concentrated with a PCR cleanup kit (QIAGEN), loaded onto a 1% agarose gel, and separated by gel electrophoresis.

Cas9-induced mutations were detected using the SURVEYOR Mutation Detection Kit (Transgenomic/IDT). Briefly, an approximately 500bp region surrounding the expected mutation site was PCR-amplified using Herculase II (600675, Agilent Technologies). PCR products were column purified (Qiagen) and subjected to a series of melt-anneal temperature cycles with annealing temperatures gradually lowered in each successive cycle ^{25 (link)}. SURVEYOR nuclease was then added to selectively digest heteroduplex DNA. Digest products were visualized on a 2% agarose gel.