LDW_BDC Muscle scRNAseq

In a given mouse, both tibialis anterior (TA) muscles were injected with 10 µl of notexin (10 µg/ml; Latoxan, France).

At various days post injury, the mouse was sacrificed and the TAs were collected. Each TA was proccessed independently to generate single cell suspensions.

Muscles were enzymatically digested with 8 mg/ml Collagenase D (Roche, Basel, Switzerland) and 10 U/ml Dispase II (Roche, Basel, Switzerland) and then manually dissociated to generate cell suspensions.

Myofiber debris was removed by filtering the cell suspensions through a 100 µm and then a 40 µm filter (Corning Cellgro # 431752 and # 431750).

After filtration, erythrocytes were removed by incubating the cell suspension in erythrocyte lysis buffer (IBI Scientific # 89135-030).

After digestion, the single-cell suspensions were washed and resuspended in 0.04% BSA in PBS at a concentration of 10⁶ cells/ml. A hemocytometer was used to manually count the cells to determine the concentration of the suspension.

Single-cell RNA-sequencing libraries were prepared using the Chromium Single Cell 3’ reagent kit v3 (10x Genomics, Pleasanton, CA) following the manufacturer’s protocol.

Cells were diluted into the Chromium Single Cell A Chip to yield a recovery of 6,000 single-cell transcriptomes with <5% doublet rate.

Libraries were sequenced on the NextSeq 500 (Illumina, San Diego, CA).

 The mice were sacrificed, and TA muscles were collected at 0, 1, 2, 3.5, 5, and 7 days post-injury. Each TA was processed independently to generate single cell suspensions.