GSM4979413: BTS_D7_1; Mus musculus; RNA-Seq
Tenotomy injury site, consisting of tissue from the Achilles' tendon insertion at the gastrocnemius to the distal tendon enthesis at the calcaneus and surrounding tissue, excluding bone and skin, was harvested 7 days after injury. Tissue samples were digested for 20 minutes in 750U/ml Type 1 Collagenase and 7U/ml Dispase II (Gibco) in Roswell Park Memorial Institute (RPMI) medium at 37°C under constant agitation of 160rpm. Digestions were subsequently quenched with 2% FBS in PBS and filtered through 40μm sterile strainers. Cells were then washed in 2% FBS in PBS, counted and resuspended at a concentration of 1000-1200 cells/ul. Cell viability was assessed with Trypan blue exclusion on a Countess II (Thermo Fisher Scientific) automated counter and only samples with >85% viability were used for scRNA sequencing. Enough reagent was taken to sequence the transcriptome of 5000 cells according to 10x guidelines. 3' Samples:  3' V3.1 Chemistry with NextGEM Chip G  NovaSeq 6000 S4 flowcell 300 cycle kit - this was loaded to target 250M reads/sample (5000 cells, 50K reads/cell), at 300pM (these were shared flowcells)  Read lengths: 150 x 8 x 150