Single-cell RNA-Seq of Scleraxis-Positive Cells

Control, non-injured Tnmd^−/−ScxGFP⁺ and WT ScxGFP⁺ Achilles tendons (n = 2) were explanted and GFP⁺ cells were isolated by collagenase digestion (8 h) and filtration according to [22 (link)]. Next, cells (n = 20/genotype) were resuspended in PBS, placed on Adcell diagnostic slides (Thermo Fisher, Waltham, Massachusetts, USA), picked up in 1 µl PBS each using a micromanipulator (Patchman NP2) with pump (CellTram, both Eppendorf, Hamburg, Germany) and subsequently stored in Smart-Seq2 lysis buffer at −80 °C. The whole transcriptome amplification (WTA) and Illumina Nextera XT library preparation (Illumina, San Diego, California, USA) were performed as described by Picelli et al. [32 (link)]. The libraries were quantified using the KAPA Library Quantification kit (Roche Diagnostics, Mannheim, Germany), pooled in equimolar amounts and sequenced paired-end with read length of 2 × 150 bp and yield of 30 million reads per library on an Illumina HiSeq. In total, six ScxGFP⁺ cells (n = 6/genotype) were subjected to scRNA-Seq. Bioinformatic analysis is described in Supplementary information.