Single cell RNA Sequencing (scRNA-Seq) of patient with ARPC5 deficiency and healthy control. Single cell RNA Sequencing (scRNA-Seq) of patient with ARPC5 deficiency and healthy control

We performed a targeted scRNA-seq approach using the BD Rhapsody™ Single Cell Analysis Systems (BD Biosciences). Peripheral blood mononuclear cells (PBMCs) were labeled with sample tags (BD Human Single Cell Multiplexing Kit) and AbSeq antibodies (CD1c, CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD18, CD19, CD20, CD21, CD23, CD24, CD25, CD27, CD28, CD31, CD38, CD40, CD44, CD45RA, CD45RO, CD56, CD62L, CD123, CD127, CD134, CD137, CD161, CD183, CD185, CD186, CD196, CD197, CD272, CD278, CD279, CD303, CD314, CD337, CD357, CD366, HLA-DR, IgD, IgM, TCRαβ, TCRγ8, Vα24-Jα18) and counted. Single-cell capture was performed using the BD Rhapsody Express following manufacturer’s instructions. The libraries were pooled and sequenced on NovaSeq 6000 S2 as a dual index pair-end run. Fastq files were processed using BD's Rhapsody analysis pipeline on the Seven Bridges Platform using default parameters, the GRCh38-PhiX-gencodev29 reference genome and the gencodev29-20181205 transcriptome annotation. Data (molecules per gene per cell based on DBEC error correction) were analyzed using BD SeqGeq v1.8 and the Seurat plug-in to perform dimensionality reduction (UMAP) and differential analysis output for selected cell subsets (CD4+ T cells, CD8+ T cells, NK cells, NKT cells, classical monocytes, intermediate monocytes, non-classical monocytes). Overall design: Peripheral blood mononuclear cells (PBMCs) were labeled with sample tags (BD Human Single Cell Multiplexing Kit) and AbSeq antibodies (BD Human Single Cell Multiplexing Kit). Single-cell capture was performed using the BD Rhapsody Express following manufacturer’s instructions. Samples were sequenced using the Illumina NovaSeq and analyzed using SeqGeq v1.8 from BD

我们采用靶向单细胞RNA测序（scRNA-seq）方法，使用BD Rhapsody™ 单细胞分析系统（BD Rhapsody™ Single Cell Analysis Systems，BD Biosciences）完成实验。对外周血单个核细胞（PBMCs）使用样本标签（BD Human Single Cell Multiplexing Kit）及AbSeq抗体（涵盖CD1c、CD3、CD4、CD5、CD8、CD11c、CD14、CD16、CD18、CD19、CD20、CD21、CD23、CD24、CD25、CD27、CD28、CD31、CD38、CD40、CD44、CD45RA、CD45RO、CD56、CD62L、CD123、CD127、CD134、CD137、CD161、CD183、CD185、CD186、CD196、CD197、CD272、CD278、CD279、CD303、CD314、CD337、CD357、CD366、HLA-DR、IgD、IgM、TCRαβ、TCRγ8、Vα24-Jα18）进行标记并计数。按照制造商操作指南，使用BD Rhapsody Express完成单细胞捕获。将测序文库混合后，在NovaSeq 6000 S2平台以双索引双端测序模式完成测序。使用BD的Rhapsody分析流程，在Seven Bridges平台上以默认参数处理Fastq文件，参考基因组采用GRCh38-PhiX-gencodev29，转录组注释采用gencodev29-20181205。基于DBEC纠错得到的每个细胞每个基因的分子计数数据，采用BD SeqGeq v1.8及Seurat插件进行分析，完成降维（UMAP）与差异分析，并输出选定细胞亚群的结果，包括CD4+ T细胞、CD8+ T细胞、NK细胞、NKT细胞、经典单核细胞、中间型单核细胞及非经典单核细胞。整体实验设计：对外周血单个核细胞（PBMCs）使用样本标签（BD Human Single Cell Multiplexing Kit）及AbSeq抗体（BD Human Single Cell Multiplexing Kit）进行标记；按照制造商操作指南，使用BD Rhapsody Express完成单细胞捕获；样本经Illumina NovaSeq平台测序后，采用BD SeqGeq v1.8完成数据分析。