GSM6647488: HLI (tdT cells); Mus musculus; RNA-Seq
Hindlimb gastrocnemius muscles were removed at 14 days after femoral artery ligation or sham operation (10 mice pre group). A single-cell suspension was prepared from hindlimb muscles by mechanical and enzymatic dissociation with minor modifications. Briefly, hindlimb muscles were dissected and minced into a fragmented muscle suspension. The muscle suspension was digested in Ham's F10 containing 400 U/ml collagenase II (Worthington) and 11 U/ml dispase (Life Technologies) at 37 ℃ with reciprocal shaking (60 rpm/min) for 1 h. Detached cells were then filtered through a 40 μm cell strainer (BD Biosciences), and red cells were removed by lysis buffer. Subsequently, the remaining cells were washed with prewarmed (37 ℃) RPMI 1640 medium containing 10% FBS by centrifugation at 500 g for 5 min and suspended in PBS. Human muscle tissues were stored in prechilled PBS. A single-cell suspension was prepared from lower limb muscles by mechanical and enzymatic dissociation with minor modifications. Briefly, the muscle was cut into small fragments, and visible fat and connective tissue were removed. Then, the samples were transferred to low-glucose Dulbecco's modified Eagle's medium (LG-DMEM) containing 0.1% collagenase D and digested in a 37 °C water bath with agitation (60–70 rpm) for 60 min. Detached cells were then filtered through a 70 μm cell strainer (BD Biosciences), and red cells were removed with lysis buffer. Subsequently, the remaining cells were washed with prewarmed (37 ℃) LG-DMEM containing 10% FBS by centrifugation at 500 g for 5 min and suspended in PBS for further experiments. Cells were then stained with a LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (1:1000) and Hoechst 33342 (Invitrogen, H3570, 1:1000) for 20 mins on ice. After being washed in PBS, cells were re-suspended in PBS. Single live cells (Hoechst+/Dead Cell Stain-) were sorted into PBS with 0.04% BSA using a BD FACS SymphonyS6 (BD Biosciences). In addition, we sorted tdTomato+ cells by the same method. A Chromium Single Cell 3' Reagent Kit v3 was used, and Standard 10X Chromium Single Cell 3' v3 (10X Genomics GemCode Technology) protocols were followed for scRNA-seq. Briefly, single cells with specific 10X barcodes and unique molecular identifiers (UMIs) were generated by partitioning the cells into Gel Bead-in-Emulsions. A cDNA library was generated and sequenced with a Novaseq (S4) 6000 instrument (PE150).