scRNA-seq of murine tendon from sham and injured group

ScRNA-seq was performed on murine single cell suspension collected  from sham and injured group. Tendon tissues were collected and dissected into pieces. Then they were digested in a mix of collagenase II (Roche, 3 mg/ml) and dispase II (Sigma-Aldrich, 4 mg/ml), prepared in DMEM for 1 hour at 37 °C. Digestions were subsequently quenched with 10% FBS DMEM and filtered through 40μm sterile strainers. Cells were then washed in PBS with 0.04% BSA, counted and resuspended at a concentration of ~1000 cells/μl. Cell viability was assessed with Trypan blue exclusion on a Countess II (Thermo Fisher Scientific) automated counter and only samples with >85% viability were processed for further sequencing.

本研究对从假手术组与损伤组采集的小鼠单细胞悬液开展了单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）。首先收集肌腱组织并剪切成小块，随后将其置于以DMEM配制的混合消化液中，于37℃下消化1小时，该消化液包含3 mg/ml的Ⅱ型胶原酶（collagenase II，Roche）与4 mg/ml的Ⅱ型分散酶（dispase II，Sigma-Aldrich）。消化完成后，使用含10%胎牛血清（fetal bovine serum, FBS）的DMEM培养基终止消化反应，并通过40μm无菌细胞筛过滤细胞悬液。随后用含0.04%牛血清白蛋白（bovine serum albumin, BSA）的磷酸盐缓冲液（phosphate-buffered saline, PBS）洗涤细胞，计数后将细胞重悬至约1000个/μl的浓度。最后采用台盼蓝排斥法，借助Countess II自动细胞计数仪（Thermo Fisher Scientific）检测细胞活力，仅选取细胞活力＞85%的样本进行后续测序。