Illumina NovaSeq 6000 paired end sequencing; Gene expression profiling of a model of CRISPR-mediated insulator disruption in the MMP locus in triple-negative breast cancer cell lines
Gene expression profiling of a model of CRISPR-mediated insulator disruption in the MMP locus in triple-negative breast cancer cell lines
Cells were collected using 0.25% Trypsin-EDTA 1x (Gibco) Cells were cultured using RPMI 1640 media complemented with 10% FBS and penicillin/streptomycin at 5% CO2 37ºC. Cells were transiently transfected with either empty pSpCas9 or pSpCas9 containing a sgRNA sequence against insulator element 8. Transfection was performed using Lipofectamine3000 following manufacturer's guidelines. Cells were then sorted using FACSAria Fusion using GFP+ as a reporter. The established models were then subcultured to perform high throughput experiments. RNA was purified using EZNA Total RNA kit I (Omega Bio-Tek) TruSeq stranded mRNA kit (Illumina) following the manufacturer's protocol.