GSM9241389: Quad muscle, subcritically injured, ADT; Mus musculus; OTHER
Entire injured (or uninjured for controls), left quadriceps were harvested and digested with 5,500U/ml collagenase II and 2.5U/ml Dispase II for 1.5 h in a shaking 37 °C water bath. The digested muscles were filtered through a cell strainer to obtain a single cell suspension. Red blood cells were lysed for 15 minutes at room temperature, samples were centrifuged, and pelleted cells were washed with cell staining buffer after supernatant was aspirated. Cells were counted so that 125,000 cells per sample were transferred to new tubes, centrifuged, and resuspended with Fc blocking antibody in cell staining buffer for 10 minutes. Cells from each sample were subsequently stained with TotalSeq-C cell hashtag reagents (BioLegend) to barcode cells from individual animals. Samples from animals in the same experimental group (n=4) were washed and then pooled to total 500,000 cells per condition (3 conditions: uninjured, subcritical, critical VML). Each pooled treatment group was stained with 25uL of reconstituted TotalSeq-C Mouse Universal Cocktail (BioLegend) in a total staining volume of 50uL for 30 minutes at 4°C. Cells were washed and resuspended to a final concentration of 1,000 cells/μL. The 10x Genomics Chromium Next GEM Single Cell 5' Reagent Kits v2 (Dual Index) protocol was followed to complete single cell preparation for sequencing (CG000331). ~10,000 cells (per 500,000 pooled condition group) were loaded onto Chromium Next GEM Chip K for Gel Beads-in-emulsion (GEM) generation. After GEM generation, Gel Beads were dissolved, and co-partitioned single cells were lysed using a thermal cycler to produce 10x Barcoded, full-length cDNA from mRNA. GEMs were broken and pooled following cDNA synthesis, and 10x Barcoded cDNA was purified and subsequently amplified according to protocol. Quality control and quantification of cDNA was analyzed on an Agilent Bioanalyzer High Sensitivity chip and Qubit fluorometer. 5' gene expression libraries were constructed from amplified cDNA, and sample indices were added to allow for simultaneous sequencing of multiple samples in accordance with 10x Genomics protocol. Post library construction quality control was validated with Agilent Bioanalyzer High Sensitivity chip and Qubit fluorometer. Samples were sequenced on an Illumina sequencer using the NextSeq Mid Output (150 cycles) kit.