Single cell profiling of mouse blood cells using CEL-seq2

Gene expression in mouse blood cells from sorted via FACS analysis into a 384-well plate were profiled using a modified CEL-seq2 protocol. Overall design: B lymphocytes (B220+ FSC-Alow), erythroblasts (Ter119+ CD44+, FSC-Amid/high), granulocytes (Mac1+ Gr1+) and high-end progenitor/stem (Lin- Kit+ Sca1+) were sorted from the bone marrow of a C57BL/6 10-13 week old female. T cells (CD3+ FSC-Alow) were isolated from the thymus of the same mouse. Bone marrow and thymus were dissociated mechanically, washed and stained with antibodies for 1hr on ice. Single cells were deposited on a 384 well plate using an Aria cell sorter (Beckman). Index data was collected and an adapted CEL-seq2 protocol used to generate a library for sequencing. The reads were sequenced by Illumina Nextseq and preprocessed by the scPipe R/Bioconductor software.

经荧光激活细胞分选术（Fluorescence-Activated Cell Sorting, FACS）分选并接种至384孔板的小鼠血细胞，其基因表达谱采用改良CEL-seq2方案完成分析。整体实验设计如下：B淋巴细胞（B220+ FSC-Alow）、成红细胞（Ter119+ CD44+，FSC-Amid/high）、粒细胞（Mac1+ Gr1+）以及高级祖细胞/干细胞（Lin- Kit+ Sca1+）均从10~13周龄雌性C57BL/6小鼠的骨髓中分选获得。T淋巴细胞（CD3+ FSC-Alow）则分离自同一只小鼠的胸腺。骨髓与胸腺经机械解离、洗涤后，于冰上与抗体共孵育1小时进行标记。使用Aria细胞分选仪（Beckman）将单细胞接种至384孔板并采集索引分选数据，随后采用适配改良的CEL-seq2方案构建测序文库，经Illumina Nextseq平台测序后，由scPipe R/Bioconductor软件完成测序reads的预处理。