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

We investigated how age and sex affected mRNA kinetics in chondrocytes isolated from young female and male horses, as well as from older females. We chose to focus on this cell type due to its critical role in articular cartilage maintenance and to the relative ease of isolating appropriate cell numbers from equine metacarpophalangeal joint to permit prompt analysis and avoid cell dedifferentiation. Chondrocytes plated at high density were analysed with SLAM-Seq, which uses the chemical conversion of 4-thiouridine (4sU) labelled RNA, allowing for the detection of newly synthesised and pre-existing RNA within one sample. We used four-hour pulse of 4sU immediately prior to RNA extraction. Based on the findings from Erhard et al., this duration is expected to provide sufficient labelling time to accurately estimate the half-life of genes ranging from approximately one to 10 hours. To increase the detection of a sufficient number of conversions per transcript, we used 500 µM 4sU and sequenced to the libraries 150bp from both ends. Our results demonstrated progressive degradation of unlabelled, pre-existing RNA and concurrent transcription of 4sU-labelled, newly synthesised RNA over time. Overall design: RNA-seq profiling of 4-thiouridine (4-sU) labeled chondrocytes isolated from young female, young male, and old female equines