Single-Cell RNA-Seq of Rat Skeletal Muscle

Cell isolations were performed as previously described in mice [37 (link)] and modified slightly for rats [28 (link)]. Briefly, the gastrocnemius muscles from WB and HS male rats were excised and placed in muscle dissociation media (MDM) (Hams F-10 (Gibco, USA), 10% Horse Serum (Thermo Fisher), 1% penicillin/streptomycin (Gibco), 800 U/ml Collagenase II (Gibco)), and minced using sterilized surgical equipment. The muscle homogenate was then incubated in MDM for 1 h at 37 °C with gentle agitation. Following incubation, samples underwent further incubation in 1000 U/ml Collagenase II (Gibco) and 11 U/ml dispase (Gibco) for 30 min at 37 °C. The single-cell suspension was passed through an 18-gauge needle approximately 10 times prior to 0.2-μm filtration. Single cells were incubated in propidium iodide to identify dying/dead cells for removal via fluorescence-activated cell sorting (Sony Biotechnology, USA). Single-cell suspensions from each group were added to a Chromium Controller (10X Genomics, USA) using the Single Cell 3’ Reagent Kit per manufacturer’s instructions and sequenced on an Illumina HiSeq platform (Novogene, USA), yielding 200 million reads/sample.