Single-cell RNA sequencing of meniscus from ACTA2-lineage traced mice, rats and dogs
Meniscal tissues were collected from: 1) Genetic Lineage Tracing Mice: ACTA2-CreERT; R26-tdTomato mice (tamoxifen-induced, 75 mg/kg, i.p. for 5 days). Menisci were harvested at 2 weeks (n=13) and 10 weeks of age (n=10). 2) Other Species: Menisci from 12-week-old male Sprague-Dawley rats (n=4) and 12-month-old male beagles (n=2). All tissues were placed in cold PBS post-dissection for immediate processing. This is encompassed within the sample collection protocol above. Menisci were surgically dissected from knee joints of euthanized animals under aseptic conditions. Tamoxifen induction: ACTA2-CreERT; R26-tdTomato mice received intraperitoneal injections of tamoxifen (75 mg/kg body weight) dissolved in corn oil, once daily for five consecutive days.  Single-cell suspensions were prepared by enzymatic digestion (e.g., collagenase) of minced meniscal tissue. The suspension was filtered, washed, and resuspended. Cell viability and concentration were assessed via trypan blue exclusion and counting. This protocol yields intact single cells; no bulk nucleic acid extraction was performed. Libraries were constructed using the BD Rhapsody system. Single cells and barcoded beads were co-encapsulated in microwells. Poly-adenylated RNA was captured, reverse-transcribed, and each cDNA was tagged with a cell label and UMI. Amplification used the BD Resolve Whole-Transcriptome Amplification kit (second-strand synthesis, adaptor ligation, PCR). Final libraries were prepared by random priming PCR to enrich 3â€˜ end fragments.