GSM7979114: FAPs_DEN-3dpi; Mus musculus; RNA-Seq
Limb muscles below knee were dissected from mice at indicated time points post either sciatic nerve crush injury or denervation at indicated time points. After removal of nerves and tendons, muscles were finely chopped with surgical scissors and mechanically dissociated in Dulbecco's modified Eagle's medium (DMEM) containing 10% horse serum (Hyclone, Logan, UT, USA), collagenase II (800 units/mL; Worthington, Lakewood, NJ, USA), and dispase (1.1 units/mL; ThermoFisher Scientific, Waltham, MA, USA) at 37°C for 40 min with mild agitation. Digested suspensions were subsequently triturated by sterilized syringe with 20G1/2 needle (BD Biosciences, Franklin Lakes, NJ, USA) and washed with DMEM to harvest mononuclear cells. Mononuclear cells were stained with anti-CD45-APC, anti-CD31-APC, anti-Ter119-PE, anti-Sca1-FITC, anti-Vcam1-biotin and streptavidin-PE/Cy7. To exclude dead cells, 7AAD (Sigma aldrich; St. Louis, MO, USA) was used prior to FACS sorting. 7AAD–Lin–Vcam–Sca1+ FAPs were isolated using a FACS Aria III cell sorter (BD Biosciences) with 4-way-purity precision. Isotype control density plots were used as a reference for positive gating. Libraries for each sample of FAPs were generated with the 10X Genomics Chromium Next GEM Single Cell 3p RNA library v3.1 kit, following the manufacturer's instructions, and a total of 5,000 cells were targeted for recovery from each sample of FAPs. Libraries were sequenced on the Illumina HiSeq X Ten following the manufacturer's protocols.