GSM7721187: SMC from mosue skeletal muscle vasculature-iSM-G5C-KO3; Mus musculus; RNA-Seq
For RNA sequencing, EGFP-expressing SMC were isolated from skeletal muscle vasculature of tamoxifen-induced control and iSM-G5C-KOs carrying the mTmG reporter strain. To do so, freshly isolated mouse skeletal muscles were chopped on ice into small pieces using surgical scissors,  digested with 0.2 mg/ml collagenase II (Worthington Biochemical Corporation, 4174) and 0.2 mg/ml dispase II (ThermoFischer, 17105041) in HBSS at 37°C for 30 min with 300 rpm shaking. By the end of the digestion procedure, cell suspensions were immediately filtered through 100 µm cell strainers, centrifuged at 1000 x g for 4 min at 4°C. Cell pellets were then suspended in cold HBSS containing 1% BSA, incubated with APC conjugated anti-CD31 (eBioscience, 17-0311), anti-CD45 (eBioscience, 17-0451-82) and anti-Ter119 (eBioscience, 17-5921) antibodies (dilution 1:100) for 15 min at 4°C to enable non-SMC depletion using anti-APC beads (Miltenyi Biotec, 130-090-855) following instructions of AutoMACS Pro Separator (Miltenyi Biotec). The negative AutoMACS fractions were centrifuged at 1000 x g for 4 min at 4°C and resulting cell pellets were suspended in 1 ml cold DMEM. With the DAPI based live/dead gating strategy, viable EGFP-positive cells were sorted into 1.5 ml reaction tubes containing cold DMEM/PBS (v/v 1:1) using BD FACSAria III or BD FACSMelody and spun down at 600 x g for 5 min at 4°C. Supernatants were removed and RNA were isolated as described above. 5ng total RNA was used as input for SMARTer® Stranded Total RNA-Seq Kit - Pico Input Mammalian Kit (Takara Bio).