GSM6528922: 40A2; Mus musculus; RNA-Seq
Mice were anesthetized via intraperitoneal injection of 2,2,2-tribromoethyl alcohol (Aldrich T4840-2) and then perfused with warm PBS.  Following removing the fascia, skeletal muscle was harvested and cut into small pieces with scissors which was then followed by enzymatic digestion with a mixture of Collagenase D (Roche; 1.5 U ml-1; # 11088882001) and Dispase II (Roche; 2.4 U ml-1; # 04942078001) for an hour at 37 °C. Samples were vortexed every 20 minutes during incubation. Digested tissues were filtered through a 40-μm cell strainer (FALCON, # 352340), washed with PBS and collected by centrifugation at 545g for 7min.  Cell preparations were blocked with PBS containing 2 mM EDTA and 2% FBS (FACS buffer) + anti-Fcy receptor antibody on ice for 10 minutes and then stained with various antibodies on ice for 15 minutes (Supplementary Table 2). Stained cells were washed for cell sorting. Cell sorting was performed using a BD FACSAria (Becton Dickinson) equipped with three lasers or BD Influx cell sorter both equipped with four lasers.  RNeasy Micro Kit (Qiagen, # 74004) was used to isolate RNA from sorted cells. 10 μ of 2β-mercaptoethanol was added to 1mL of the buffer RLT (lysis buffer) before use. Sample quality control was performed using the Agilent 2100 Bioanalyzer. Qualifying samples were then prepped following the standard protocol for the NEBnext Ultra ii Stranded mRNA (New England Biolabs). Paired-end sequencing was performed on the Illumina NextSeq 2000 using the P3 100 cycle Kit (Illumina).