GSM6528917: FR121_macrophages; 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). Finally, cells were washed and stained with cell hashing antibodies against mouse CD45 and MHC class I (Biolegend, # 155801, # 155803, # 155805, # 155807) to pool up to four samples for one single-cell analysis run. Cell sorting was performed using a BD FACSAria (Becton Dickinson) equipped with three lasers or BD Influx cell sorter both equipped with four lasers. Cells were sorted twice. Viable cells were delivered into a Chromium Controller (10X Genomics), captured and libraried with the Chromium single cell 3' reagent kit v3 (10X Genomics) as per the manufacturer's protocol. cDNA libraries were sequenced on Illumina Nextseq 500 using the High Output 75 Cycle kits to a read depth of 65,000 mean reads per cell.