title: RNaseq of primary human OA chondrocytes alones or in transwell cocultures with primary human Adipose-derived Mesenchymal Stromal Cells, in presence of 10nM rapamycin or DMSO as vehicle control
library_construction_protocol: Primary human chondrocytes were extracted from OA cartilage following total knee replacement surgery. Cartilages were sliced, washed, digested with Pronase E for 1h and then digested with Collagenase II overnight. The next day, chondrocytes were seeded at 3x10^5 cells/cm² in flasks in DMEM, 10%FBS, 1% L-Gln , 1%PS and were allowed to stand for 15 to 21 days, depending on proliferation rate of the donor. P0 OA chondrocytes were then seeded at 6x10^4 cells/cm² for 1 week and P1 OA chondrocytes were then stored in liquid nitrogen before cocultures. Total RNA was isolated using mini RNeasy kit (Qiagen) with an on-column DNAse I digestion step, according to the manufacturer instructions.The quantity and quality of extracted RNA was evaluated using the NanoDrop One spectrophotometer and the Agilent 2100 BioAnalyzer. Libraries were constructed by the Beijing Genomics Institute (BGI) on a DNBSEQ Eukaryotic Stranded Transcriptome library preparation for Stranded mRNA Library. All RNA integrity numbers were above 9.