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 Overall design: 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).

本研究分析了年龄对关节组织中多聚腺苷酸化位点（polyadenylation site）使用模式的影响。我们从关节软骨（articular cartilage）或滑膜（synovium）中纯化得到总RNA，随后开展QuantSeq-Rev 3'端RNA测序，以鉴定所有转录本所利用的多聚腺苷酸化位点。该3'端测序方法从转录本的多聚腺苷酸尾（polyA tail）起始，沿3'至5'方向进行测序，因此每条测序读段均可对应一个多聚腺苷酸化位点。随后我们使用PolyAminer软件，并对其代码进行适配改造，以识别并屏蔽基因组多聚腺苷酸区域，确保这些区域在测序读段比对（read alignment）后可参与位点比较分析。我们发现有证据表明，年龄会影响这两种组织中的多聚腺苷酸化位点使用模式。
整体实验设计：将年轻和老年马匹的冷冻组织样本使用Braun Mikrodismembrator进行匀浆处理，随后用TRIzol（Thermo Fisher Scientific, Waltham, MA, USA, #15596018）进行总RNA提取。加入氯仿并完成相分离后，吸取水相层，通过RNeasy迷你试剂盒（Qiagen, Manchester, UK, #74104）进行纯化，过程包含柱上DNase消化步骤。文库制备采用QuantSeq-Reverse方法，NextSeq 75循环高通量测序搭配V2化学试剂盒的测序工作由Lexogen（Vienna, Austria）完成。