Gene expression profiles of U2OS human osteosarcoma cell lines with ablation of NR1D1 or NR1D2 gene. 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-ERBα or REV-ERBβ (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.