RNA-seq of OA knee cartilage
Total RNA was mixed with 1l of anchored oligo-dT primer and 1 l of dNTP mix (10 mM, Fermentas), denatured at 72 C for 5 min. Seven microliters of the first-strand reaction mix (containing 0.50 l SuperScript II reverse transcriptase (Invitrogen), 0.25 l RNAse inhibitor (Clontech), 2 l Superscript II First-Strand Buffer (Invitrogen), 0.25 l DTT (Invitrogen), 1 l betaine (Sigma), 0.9 l MgCl2 (Sigma), and 0.1 l nuclease-free water (Gibco)) were added to each sample. Reverse transcription reactions were carried out at 42 C for 90 min, followed by 10 cycles of (50 C for 2 min, 42 C for 2 min). The reverse transcriptase was inactivated at 70 C for 15 min.Second strand synthesis was carried out after adding 2 l 10X second strand synthesis buffer, 1 l Enzyme mix, 100ng  cDNA and water to a final reaction volume of 20 l. The reaction was incubated at 16 C for 150 min and then purified with AMPure XP beads.