Gene expression profiles of U2OS human osteosarcoma cell lines with ablation of NR1D1 or NR1D2 gene
We have recently established a panel of mutant U2OS human osteosarcoma cells lacking either REV-ERBa or REV-ERBß (encoded by NR1D1 and NR1D2 genes, respectively) expression by a CRISPR/Cas9 and dual sgRNA-mediated gene deletion strategy. We then intended to dissect redundant and isoform-specific roles of these circadian nuclear receptors in controlling their target genes by comparing transcriptome profiles among wild-type and mutant U2OS cells. Overall design: U2OS (ATCC HTB-96) cells were mainly cultivated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic solution in a humidified incubator containing 5% CO2, at 37°C. To generate mutant cell lines, U2OS cells were transfected with 2 µg of an sgRNA expression vector by use of the Lipofectamin 3000 Transfection Reagent. After 48 h of transfection, cells were selected in the presence of 2 µg/ml puromycin. Cellular colonies were then individually transferred onto 24-well culture plates and deletion of intended genomic regions were confirmed by PCR-based genotyping. A cell line with truncations in both NR1D1 and NR1D2 genes were produced by a sequential transfection of sgRNA-expressing plasmids.