GSM5350805: CD45e_young_1wk_Naive; Mus musculus; RNA-Seq
Tissue samples were obtained by cutting the quadriceps femoris muscle from the hip to just above the knee. Tissues were finely diced manually with standard razor blades and digested for 45 min at 37oC with 1.67 Wünsch U/mL (0.5 mg/mL) Liberase TL (Roche Diagnostics, Sigma Aldrich) and 0.2 mg/mL DNase I (Roche Diagnostics, Sigma Aldrich) in RPMI-1640 medium supplemented with L-Glutamine and 15 mM HEPES (Gibco). The digested tissues were then pestled through 70 μm cell strainers (ThermoFisher Scientific) with RPMI-1640 (supplemented as before), and then washed twice with 1X DPBS. A Percoll (GE Healthcare) density gradient centrifugation was used to enrich the leukocyte fraction and remove blood and debris from the muscle samples and centrifuged at 2100 x g for 30 min with the lowest acceleration and no brake. Cells were washed 1X DPBS, stained with a viability live/dead amine reactive dye, washed with 1X DPS, blocked with anti-mouse TruStain FcX (BioLegend), and surface stained (Supplementary Table 6). Flow cytometry was performed using an Attune NxT Flow Cytometer (ThermoFisher Scientific) with a Violet, Blue, Green, and Red laser configuration. All subsequent analyses were performed on Live singlet cells using Windows based FlowJoTM software v10 (Benton Dickinson), license supplemented courtesy of the Johns Hopkins Bloomberg Flow Cytometry and Immunology Core. According to Dropseq and 10X respectively