Target Resequencing of human COL6A1 post CRISPR/Cas9 editing (Data set 2 of 2)
Normal control and patient dermal fibroblast cell lines were established from skin biopsies. Cells were nucleofected with a Cas9-GFP plasmid (Addgene #48138, courtesy of Dr. Feng Zhang) that included gRNA sequences (gRNA-A or gRNA-B), or no gRNA (No gRNA). After 48 hours, cells were collected in cell sorting medium containing 1g/mL of 4,6-Diamidino-2-phenylindole dihydrochloride (DAPI). Cells were next sorted at the NINDS Flow and Imaging Cytometry Core Facility on the MoFlo Astrios Cell Sorter. The strategy for gating was to first exclude dead cells using 1g/mL DAPI staining, and then sort on viable DAPI-negative cells that also expressed GFP. Threshold for GFP expression was established using untransfected cells as controls. GFP+ cells were sorted in complete culture medium for DNA extraction. Following cell sorting, cells were pelleted by centrifugation at 10,000xg for one minute and washed once with PBS before DNA extraction. DNA was isolated from samples using the Puregene kit (Qiagen) according to instructions. DNA library for targeted re-sequencing was prepared using two rounds of amplification. A first PCR round was prepared from genomic DNA using the KAPA HiFi HotStart PCR Kit. The forward primers for this reaction (5-ctccttggcccaaatcctat-3') were each extended in 5 with a nucleotide stagger, a barcode unique to each sample, and the Illumina forward extension (5-acactctttccctacacgacgctcttccgatct-3'), while the reverse primers (5-tagtgctgtgcaaggctgag-3') were also extended with a nucleotide stagger, a barcode unique to each sample, and the Illumina reverse extension (5- gtgactggagttcagacgtgtgctcttccgatct-3'). A second round of amplification was performed, using PCR products from the first round, using the same conditions as above, except for the number of cycles which was increased to 25. The primers used for that second round were the Illumina adapter primers forward (5-aatgatacggcgaccaccgagatctacactctttccctacacgac-3') and reverse (5-caagcagaagacggcatacgagatgtgactggagttcagacgtgt-3'). Reaction duplicates were pooled and purified with the QIAquick PCR Purification Kit (Qiagen).