Illumina HiSeq 4000 paired end sequencing; Circadian time series RNAseq in mouse primary chondrocytes
Circadian time series RNAseq in mouse primary chondrocytes
Primary chondrocytes from 5 day old mice were isolated as follows. 5 day old C57bl/6 PER2::Luc mice were sacrificed by decapitation. Knee, hip and shoulder joints were dissected, and any soft tissue removed. Joint cartilage was subjected to pre-digestion with collagenase D (3 mg/mL) in DMEM two times for 30 min at 37°C with intermittent vortex to remove soft tissue leftovers. Subsequently, cartilage was diced using a scalpel and digested overnight at 37°C. Cells were dispersed by pipetting and passed through 70 μM cell strainer. Cell suspension was then centrifuged and the pellet was re-suspended in DMEM/F12 with 10% FBS and plated in T75 flasks. Cells were passaged only once before experiments. Mouse primary chondrocytes were passaged into 6-well plates. Upon reaching confluency, media were changed to fresh and ~30 hr later osmolarity was increased by +200 mOsm using sorbitol. T0 sample was collected just before the addition of sorbitol. Then samples were collected every 4 hr for 48 hr. The experiment was repeated three times for replicates A,B and C. mRNA was extracted using RNeasy micro kit (Qiagen) according to the manufacturer's protocol. Quality and integrity of the RNA samples were assessed using a 2200 TapeStation (Agilent Technologies). Libraries were generated by the Genomic Technologies Core Facility using the TruSeq® Stranded mRNA assay (Illumina, Inc.) according to the manufacturer's protocol.