GSM6996260: DBA2/J Day 8 after CTX muscle injury (stRNA-seq); Mus musculus; OTHER
After tissue digestion and bead selection, CD45+ single-cell sorted suspensions were washed and resuspended in 0.04% BSA in PBS at a concentration of at least 400 cells/μL. Cells were counted manually with a hemocytometer to determine their concentration. Single-cell RNA-sequencing libraries were then prepared using the Chromium Single Cell 3' reagent kit v3.1 (10X Genomics, Pleasanton, CA) in accordance with the manufacturer's protocol. Briefly, the cells were diluted into the Chromium Single Cell A Chip as to yield a recovery of ~10,000 single-cell transcriptomes with < 5% doublet rate. Following the library preparation, the libraries were sequenced on the NovaSeq 6000 sequencer (Illumina, San Diego, CA) to produce about 450 million reads per library and on average a minimum of 40,000 reads per single cell. For spatial transcriptomics: fresh frozen skeletal muscle samples were cryosectioned (Leica CM1950). 10-µm sections were placed on the pre-chilled Optimization slides (Visium, 10X Genomics, PN-1000191), and the optimal lysis time was determined. The tissues were treated as recommended by 10X Genomics and the optimization procedure showed an optimal permeabilization time of 30 min of digestion and release of RNA from the tissue slide. Spatial gene expression slides (Visium, 10X Genomics, PN-1000185) were used for spatial transcriptomics following the Visium User Guides, and whole slide images were taken using a 20X objective of a Leica Aperio Versa scanner. Next-generation sequencing libraries were prepared according to the Visium user guide. Libraries were loaded at 300 pM and sequenced on a NovaSeq 6000 System (Illumina) as recommended by 10X Genomics.