title: GSM5588472: Old RE + M; Rattus norvegicus; RNA-Seq
library_construction_protocol: Live mononuclear cells were isolated from the right gastrocnemius muscle as previously described (Liu et al., 2015). Briefly, right gastrocnemius muscle was immersed in wash media (WM; Ham's F-10 (Gibco, Gaithersburg, Maryland) + 10% Horse serum (Thermo Fisher, Florence, KY) and 1X penicillin/streptomycin (Gibco)) immediately after dissection and minced using sterilized instruments. The minced muscle homogenate was incubated in muscle dissociation media (MDM; WM + 800 U/ml Collagenase II (Gibco)) at 37°C for 1 hr with agitation. The homogenate was pelleted by centrifugation (500g, 5 min) and incubated in fresh MDM supplemented with dispase (11 U/ml; Sigma, St. Louis, MO) at 37°C for 30 min with agitation. The single cell suspension was pelleted by centrifugation (500 g, 5 min), passed through 70- and 40-mm Nylon cell strainers to remove non-cellular material, and placed in a fluorescence-activated cell sorting (FACS) tube containing propidium iodide (PI) and WM to identify dead cells. Isolation of live mononuclear cells was performed by gating out cellular debris using forward/side scatter and with/without PI using an iCyt FACS (Sony Biotechnology, Champaign, IL, USA). The mononuclear cell pellet was washed and resuspended in PBS with 0.04% BSA in accordance with 10X Genomics recommendations (10X Genomics, Pleasanton, CA, USA). Cells were loaded in the 10X Genomics Chromium Controller to target capture of approximately 10,000 cells per lane using the Single Cell 3' reagent kit per the manufacturer's instructions. Libraries were made using version 3.0 chemistry and sequenced on an Illumina HiSeq platform (Novogene, Sacromento, CA), yielding a minimum 200 million reads per sample.