Illumina HiSeq 1000 sequencing; Ribosomal RNA 2'-O-methylation profile of human articular chondrocytes exposed to an osteoarthritic environment
Ribosomal RNA 2'-O-methylation profile of human articular chondrocytes exposed to an osteoarthritic environment
Non-osteoarthritic human articular chondrocytes were plated at 30 000 cells/cm2 and exposed to 20% (v/v) OA-SF (pool of 14 patients, equal volume ratios), or 20% (v/v) 0.9% NaCl for 14 days. The experiment was performed in biological replicates (5 donors). The medium was refreshed every other day.  Cells were cultured in DMEM/F-12 low glucose with GlutaMAX (Thermo Fisher Scientific, 31331-028) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich, F7524), 1% A-A (Thermo Fisher Scientific, 15240-062), and 1% nonessential amino acids (Thermo Fisher Scientific, 11140-035) at 37°C in a humidified atmosphere containing 5% CO2. Cells were exposed to 20% (v/v) OA-SF (pool of 14 patients, equal volume ratios), or 20% (v/v) 0.9% NaCl for 14 days. Cells were exposed to 20% (v/v) OA-SF (pool of 14 patients, equal volume ratios), or 20% (v/v) 0.9% NaCl for 14 days.  Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen,74134) following the manufacturer's protocol. RNA quality was determined with a 2100 Bioanalyzer (Agilent) and RNA 6000 Nano Kit (Agilent, 5067-1511). All samples had RNA integrity (RIN) number greater than 9, which was considered sufficient for further analysis.  Total RNA was randomly fragmented by alkaline hydrolysis, RNA fragments were end-repaired and converted to library using NEBNext Small RNA Library kit.