GSM5208913: SAT_PBS scRNA-seq; Mus musculus; RNA-Seq
Skeletal muscles were harvested from hind limbs of mice from PBS or Doxo or Doxo+ABT experimental groups. Skeletal muscles from 5 mice were combined for each experimental group. For skeletal muscle dissociation, a kit from Miltenyi Biotech was used (cat#130-098-305). The muscles were chopped into 2-4 mm pieces and enzymatic digestion was performed as described in the product datasheet using as recommended kit enzymes, C Tubes, (Company: Miltenyi, Cat# 130-096-334), and gentleMACS Octo Dissociator with Heaters (cat# 130-096-427). After digestion of the muscle samples, Debris Removal Solution (Company: Miltenyi, cat#130-109-398) was used to achieve cleaner single-cell suspension of muscle tissues. The single cell suspension was stained with CD31, CD45, Sca1, a7, and PDGFRa for FACS (FACS Aria II, BD Bioscieneces). FAPs were identified as Sca1+, PDGFra+, CD31-, CD45- and a7-integrin- and SCs were identified as a7-integrin+, Sca1-, PDGFra-, CD31-, and CD45- {Joe, 2010 #46;Liu, 2015 #47}. Nuclei were isolated from the biopsies using the Singulator instrument (S2 Genomics).  The instrument was primed with cold nuclei isolation and storage buffers (S2 Genomics) and the biopsy was loaded into the cartridge and covered with the 19.7 mm grinding cap.  The “Nuclei_All_Tissues” protocol was used to isolate nuclei, after which the nuclei were centrifuged for 5 minutes and resuspended in buffer.  The nuclei were counted using a Countess II Automated Cell Counter (Invitrogen), centrifuged again for 5 minutes and resuspended at the proper concentration for use in the Chromium Single Cell 5' Library and Gel Bead Kit v1 (10X Genomics). The FAP cells and SCs were isolated using FACS. Cell samples were then prepared for 10X single-cell RNA seq protocol to target 10K cells. The 3' single-cell RNA seq library preparation was performed by following 10X kit protocol. The QC for cDNA and libraries were performed using Tapestation according to the 10X protocol. Sequencing of the resultant libraries was then performed on Illumina sequencers as per manufacturers protocols. Samples were processed using the Chromium Single Cell A Chip Kit and Chromium Controller (10X Genomics).  Quality control was performed on the Tapestation 4200 (Agilent Technologies).  Libraries were sequenced in one lane of a NovaSeqS4 by the U.C. Davis Genomics Core.