RT Journal Article SR Electronic T1 RNASeq analysis of a Pax3-expressing myoblast clone in-vitro and effect of culture surface stiffness on differentiation JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.09.05.459022 DO 10.1101/2021.09.05.459022 A1 Louise Richardson A1 Dapeng Wang A1 Ruth Hughes A1 Colin A Johnson A1 Michelle Peckham YR 2021 UL http://biorxiv.org/content/early/2021/09/05/2021.09.05.459022.abstract AB Skeletal muscle satellite cells cultured on soft surfaces (12kPa) show improved differentiation than cells cultured on stiff surfaces (approximately 100kPa). To better understand the reasons for this, we performed an RNA-Seq analysis for a single satellite cell clone (C1F) derived from the H2kb-tsA58 immortomouse, which differentiates into myotubes under tightly regulated conditions (withdrawal of γ-interferon, 37°C). As expected, the largest change in overall gene expression occurred at day 1, as cells switch from proliferation to differentiation. Surprisingly, further analysis showed that proliferating C1F cells express Pax3 and not Pax7, confirmed by immunostaining, yet their subsequent differentiation into myotubes is normal, and enhanced on softer surfaces, as evidenced by significantly higher expression levels of myogenic regulatory factors, sarcomeric genes, enhanced fusion and improved myofibrillogenesis. Levels of RNA encoding extracellular matrix structural constituents and related genes were consistently upregulated on hard surfaces, suggesting that a consequence of differentiating satellite cells on hard surfaces is that they attempt to manipulate their niche prior to differentiating. This comprehensive RNASeq dataset will be a useful resource for understanding Pax3 expressing cells.Competing Interest StatementThe authors have declared no competing interest.