title: Illumina HiSeq 4000 paired end sequencing; Gene expression profile of TGFβ3 stimulation and TRPV4 activation/inhibition in the TC28a2 human chondrocyte cell line
design: Gene expression profile of TGFβ3 stimulation and TRPV4 activation/inhibition in the TC28a2 human chondrocyte cell line
library_construction_protocol: TC28a2 cells were shared by Dr Louise Reynard (Newcastle University, UK). TC28a2 chondrocytes were plated onto 6-well plates at a density of 150,000 cells per well and grown in DMEM+10%FCS for 24 h, then serum starved overnight.  Cells were stimulated with 10 ng/mL TGFβ3, and/or 100 nM GSK101 for TRPV4 activation, and/or 500 nM GSK219 for TRPV4 inhibition as appropriate for each condition. GSK101 was always added 15 min after TGFβ. GSK219 was added along with TGFβ3 (15 min before GSK101). Cells were lysed and total RNA isolated 4 h post TGFβ stimulation (3 h 45 m post GSK101). Cells were lysed and total RNA isolated 4 h post TGFβ stimulation (3 h 45 m post GSK101) using the mirVana isolation kit (Thermofisher), according to the manufactures protocol for total RNAs. Library preparation of samples was performed using the Illumina TruSeq Stranded mRNA Sample Preparation Kits (Illumina).