Characterising the effect of age and sex on post-transcriptional regulation in synovial joint tissues

Our analysis determined the effect of age on polyadenylation site usage in joint tissues. Using total RNA purified from articular cartilage or synovium, we performed QuantSeq-Rev 3’ end RNA sequencing to identify polyadenylation sites utilised in all transcripts. This 3’ end sequencing method reads 3’ to 5’, starting in the polyA tail of the transcript, and so every read identifies a polyadenylation site. We then utilised PolyAminer software, with adaptations made to the code to identify and mask genomic polyA regions so that they would contribute to the site comparisons following read alignment. We found evidence that age affected polyadenylation site usage in both of these tissues Frozen tissue samples from young and old horses were homogenised using a Braun Mikrodismembrator and extracted in TRIzol (Thermo Fisher Scientific, Waltham, MA, USA, #15596018). Following the addition of chloroform and phase separation, the aqueous layer was removed and processed through RNeasy mini kits (Qiagen, Manchester, UK, #74104), including on-column DNase digestion. QuantSeq-Reverse library preparation and NextSeq 75 cycle[M1] [TS2] High Output sequencing with V2chemistry was performed by Lexogen (Vienna, Austria).