alias: gRNA_3A
title: Amplicon-seq post CRISPR/Cas9 editing
design: Lymphoblatoid cell lines were grown in suspension using RPMI 1640 with 15% FBS in T-75 flasks, cells were maintained between 200,000-800,000 cells/ml. 4 million cells were transfected with 33.3 g of a CRISPR/Cas9 plasmid, with or without  0.4 nmol of ssODN template, using the Lonza Cell Line Nucleofection Kit V solutions at T-020 settings on an Amaxa nucleofector II machine. Transfected cells were incubated in 2mL of medium for 24 hours, after which medium was replaced. Cells were sorted after another 24 hours. Briefly, cells were pelleted, washed twice, and resuspended in sorting buffer (Hanks balanced salt solution buffer supplemented with 1% FBS, 100 units/mL penicillin, and 100 mg/mL streptomycin). Cell suspensions were submitted to cell analysis and sorting in a FACSAria II cytometer (BD Biosciences) to isolate GFP-positive cells. Cells were then pelleted and resuspended with QuickExtract DNA Extraction Solution (Epicentre; .5mL per 104 cells). DNA was then extracted by vortexing for 15 seconds, followed by incubation at 65C for six minutes, another 15-second vortex, and finally, a two minute incubation at 98C. DNA was then concentrated by ethanol precipitation.
identifiers.namespace: SUB4326006
identifiers.id: gRNA_3A