3' droplet-based single cell RNA sequencing of a mouse large intestinal epithelium

Using 3' droplet-based single-cell sequencing, we performed the transcriptional profiling of mouse large intestinal epithelial cells at the single-cell level. For scRNA-seq of the mouse colonic epithelium, we isolated mouse colonic crypts from three female 12-week-old C57BL/6 mice. For purification of epithelial cells and removal of immune cells and dead cells, the cells were suspended in 200 ml of cold PBS and stained with 5 ml of APC anti-mouse CD326 (EpCAM) antibody (118213, BioLegend), PE anti-CD45 antibody (BD Biosciences, 1:500) and DAPI (1:1000) for 20 min. Live CD45- EpCAM+ single cells were sorted into a 1.5 mm tube using a cell sorter.本研究采用3'端液滴法单细胞测序（3' droplet-based single-cell sequencing），对小鼠大肠上皮细胞开展单细胞水平的转录组谱分析。针对小鼠结肠上皮的单细胞RNA测序（scRNA-seq）实验，我们从3只12周龄雌性C57BL/6小鼠体内分离得到小鼠结肠隐窝。为纯化上皮细胞并去除免疫细胞与死细胞，我们将细胞重悬于200ml预冷磷酸盐缓冲液（PBS）中，使用5ml APC标记抗小鼠CD326（上皮细胞黏附分子EpCAM）抗体（货号118213，BioLegend公司）、PE标记抗CD45抗体（BD Biosciences，稀释比例1:500）与DAPI（稀释比例1:1000）孵育染色20分钟。随后通过细胞分选仪将活的CD45阴性、EpCAM阳性的单细胞分选至1.5 mL离心管中。