GSM4563825: day 0 control isolated nuclei; Mus musculus; ATAC-seq
Burn and tenotomy was performed as previously described (PMID 26274052) in Hoxa11CreERt2 +/- TdTom/tom mice. Mice were allowed to ambulate normally or had their injured hindlimb immilized.Tenotomy injury site was harvested from Day 0 (no injury baseline), Day 7 mobile (normal ambulation), Day 7 immobile, and Day 42 mobile mice. 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. 100,000 remaining cells were used for nuclei isolation according to the 10X Nuclei Isolation for Single Cell ATAC Sequencing protocol (available on their website). 3' Samples: 3' V3.1 Chemistry with NextGEM Chip G NovaSeq 6000 S4 flowcell 300 cycle kit - this was loaded to target 500M reads/sample (5000 cells, 100K reads/cell), at 300pM (these were shared flowcells) Read lengths: 150 x 8 x 150 ATAC: ATAC V1.1 Chemistry with NextGEM Chip H NovaSeq 6000 SP flowcell 100 cycle kit (This was the full flowcell just for you) Read lengths: 50 x 8 x 16 x 49