Single-cell RNA sequencing of Blood-infiltrating HSC-derived phagocytes of Ms4a3Ai14 using 10X Genomics platform. IFN-γ and GM-CSF control complementary differentiation programs in the monocyte to phagocyte transition during neuroinflammation.

<strong>Single-cell RNA sequencing of Blood-infiltrating HSC-derived phagocytes of <em>Ms4a3</em>^Ai14 at onset and peak EAE using 10X Genomics platform.</strong> The sorted cells were loaded into 10x Genomics Chromium in parallel. Libraries were prepared as per the manufacturer's protocol (Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 protocol) and sequenced on an Illumina NovaSeq sequencer according to 10X Genomics recommendations (paired-end reads, R1=28, i7=8, R2=91) to a depth of around 50,000 reads per cell. Initial processing was done using Cell Ranger (v3.1.0) mkfastq and count (reads were aligned to GENCODE reference build GRCm38.p6 Release M23 with added tdTomato sequence for the dataset from <em>Ms4a3</em>^Ai14 mouse and collapse UMIs). Starting from the filtered gene-cell count matrix produced by CellRranger's in-built cell calling algorithms, we proceeded with Seurat v4 workflow.<strong>采用10X Genomics平台，对处于实验性自身免疫性脑脊髓炎（Experimental Autoimmune Encephalomyelitis, EAE）发病期与峰值期的<em>Ms4a3</em>^Ai14小鼠血液浸润性造血干细胞（Hematopoietic Stem Cell, HSC）来源吞噬细胞进行单细胞RNA测序</strong>。分选后的细胞被平行上样至10X Genomics Chromium系统。按照厂商官方方案（即Chromium Next GEM单细胞3'端试剂试剂盒v3.1流程）制备文库，并依照10X Genomics官方建议，在Illumina NovaSeq测序仪上开展测序：采用双端测序模式，设置R1读长28 bp、i7索引8 bp、R2读长91 bp，每个细胞的测序深度约为50000条reads。初始数据分析采用Cell Ranger（v3.1.0）的mkfastq与count模块完成：将reads比对至GENCODE参考基因组GRCm38.p6 Release M23，针对来自<em>Ms4a3</em>^Ai14小鼠的数据集，额外添加tdTomato序列，并对唯一分子标识符（Unique Molecular Identifier, UMI）进行去重折叠。以Cell Ranger内置的细胞识别算法生成的过滤后基因-细胞计数矩阵为起始数据，我们采用Seurat v4分析流程完成后续分析。