GSM9685898: DysfA6 mouse rep 2 (4 month old, female); Mus musculus; RNA-Seq
Quadriceps, abdominal, and paraspinal muscles were freshly harvested from 4-month-old female Dysf and DysfA6 (n=2 each, 4 mice total), washed in sterile-filtered DPBS with calcium and magnesium (MT21030CV; Thermo Fisher Scientific), combined and weighed. The tissues were minced in 6 mg/mL of collagenase type II (LS004177; Worthington) solution in 1:5 weight to volume ratio and enzymatically digested at 37°C for 1.5 hours with shaking.  The digested tissues were then filtered through 70µm nylon filter and washed with calcium and magnesium-free DPBS (14190144; Thermo Fisher Scientific).  Debris was removed from the samples using Debris Removal Solution (130-109-398; Miltenyi Biotec) following the manufacturer's instructions. Subsequent cell pellet was resuspended in 1X TheraPEAK ACK lysing buffer (BP10-548E; Lonza) to remove the red blood cells, filtered through 40µm nylon filter, and resuspended in 1% BSA/DPBS solution.  The final cell suspension was diluted 1:10, mixed 1:1 with ViaStain AOPI Staining Solution (CS2-0106-5ML; Revvity) and counted on Nexcelom Cellometer Auto2000 to ensure viability > 70%. Mononuclear cells isolated as described above were analyzed using Nexcelom Cellometer Auto2000 with AOPI fluorescent staining method for cell number and viability.  29,000 cells were loaded into the Chromium iX Controller (PN-1000328, 10X Genomics) on a Chromium GEM-X Single cell 3' Chip Kit v4 (PN-1000215; 10X Genomics), and processed to generate single cell gel beads in the emulsion (GEM) according to the manufacturer's protocol.  The cDNA and library were generated using the GEM-X Single cell 3' Kit v4 (PN-1000691; 10X Genomics) and Dual Index Kit TT Set A (PN-1000215; 10X Genomics) according to the manufacturer's manual.  Quality control for constructed library was performed by Agilent Bioanalyzer High Sensitivity DNA kit (5067-4626; Agilent Technologies) and Qubit DNA HS assay kit for qualitative and quantitative analysis, respectively. The multiplexed libraries were pooled and sequenced on Illumina Novaseq X Plus sequencer with 100 cycle kits using the following read length: 28 bp Read1 for cell barcode and UMI, and 90 bp Read2 for transcript.  The single cell library preparation and sequencing was done at Northwestern University NUseq facility core with the support of NIH Grant (1S10OD025120).