Single cell RNA sequencing of mouse brain hematopoietic cells after brain ischemia model

We performed scRNA-seq of brain CD45+ hematopoietic cells from the brain of Cd300a-fl/fl and Cd300a-fl/fl Lys2-Cre mice 0, 1 and 3 hrs after the reperfusion of middle cerebral artery occlusion (MCAO). Brain PI-CD45+ hematopoietic cells were sorted by flow cytometry from Cd300a-fl/fl and Cd300a-fl/fl Lys2-Cre mice 0, 1 and 3 hrs after the reperfusion of MCAO. Three brains were pooled per each sample. Re-staining of PI of sorted samples were measured by flow cytometry, and the viability of all samples were more than 85%. Single cells were processed through the Chromium Single Cell Platform using the Chromium Single Cell 3’ Library and Gel Bead Kit v3 (10X Genomics, PN-1000092) and the Chromium Single Cell B Chip Kit (10X Genomics, PN-1000074) as per the manufacturer’s protocol. The sequencing of libraries was performed at Macrogen Japan (Kyoto, Japan) using an Illumina HiSeq X. Processed files were generated from Cell Ranger (10X Genomics) pipeline.

我们对大脑中动脉闭塞（middle cerebral artery occlusion, MCAO）再灌注后0、1、3小时的Cd300a-fl/fl及Cd300a-fl/fl Lys2-Cre小鼠脑组织中的CD45阳性造血细胞开展了单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）。具体实验步骤如下：首先通过流式细胞术（flow cytometry）从上述基因型小鼠MCAO再灌注后0、1、3小时的脑组织中分选碘化丙啶阴性（PI⁻）CD45阳性造血细胞；每个样本混合3个小鼠的脑组织。随后对分选后的样本进行碘化丙啶复染，并通过流式细胞术检测细胞活性，所有样本的细胞活率均高于85%。接着依照厂商说明书的操作流程，使用Chromium单细胞3’端文库与凝胶磁珠试剂盒v3（10X Genomics, PN-1000092）及Chromium单细胞B型芯片试剂盒（10X Genomics, PN-1000074），通过Chromium单细胞平台（Chromium Single Cell Platform）处理单细胞样本。文库测序由位于日本京都的Macrogen日本公司采用Illumina HiSeq X测序平台完成。最终的分析处理文件通过10X Genomics的Cell Ranger（Cell Ranger）分析流程生成。