Table_1_Reference Transcriptomes of Porcine Peripheral Immune Cells Created Through Bulk and Single-Cell RNA Sequencing.XLSX

Pigs are a valuable human biomedical model and an important protein source supporting global food security. The transcriptomes of peripheral blood immune cells in pigs were defined at the bulk cell-type and single cell levels. First, eight cell types were isolated in bulk from peripheral blood mononuclear cells (PBMCs) by cell sorting, representing Myeloid, NK cells and specific populations of T and B-cells. Transcriptomes for each bulk population of cells were generated by RNA-seq with 10,974 expressed genes detected. Pairwise comparisons between cell types revealed specific expression, while enrichment analysis identified 1,885 to 3,591 significantly enriched genes across all 8 cell types. Gene Ontology analysis for the top 25% of significantly enriched genes (SEG) showed high enrichment of biological processes related to the nature of each cell type. Comparison of gene expression indicated highly significant correlations between pig cells and corresponding human PBMC bulk RNA-seq data available in Haemopedia. Second, higher resolution of distinct cell populations was obtained by single-cell RNA-sequencing (scRNA-seq) of PBMC. Seven PBMC samples were partitioned and sequenced that produced 28,810 single cell transcriptomes distributed across 36 clusters and classified into 13 general cell types including plasmacytoid dendritic cells (DC), conventional DCs, monocytes, B-cell, conventional CD4 and CD8 αβ T-cells, NK cells, and γδ T-cells. Signature gene sets from the human Haemopedia data were assessed for relative enrichment in genes expressed in pig cells and integration of pig scRNA-seq with a public human scRNA-seq dataset provided further validation for similarity between human and pig data. The sorted porcine bulk RNAseq dataset informed classification of scRNA-seq PBMC populations; specifically, an integration of the datasets showed that the pig bulk RNAseq data helped define the CD4CD8 double-positive T-cell populations in the scRNA-seq data. Overall, the data provides deep and well-validated transcriptomic data from sorted PBMC populations and the first single-cell transcriptomic data for porcine PBMCs. This resource will be invaluable for annotation of pig genes controlling immunogenetic traits as part of the porcine Functional Annotation of Animal Genomes (FAANG) project, as well as further study of, and development of new reagents for, porcine immunology.猪是极具价值的人类生物医药模型，同时也是支撑全球粮食安全的重要蛋白质来源。本研究已在批量细胞类型和单细胞两个层面解析了猪外周血免疫细胞的转录组。首先，通过细胞分选从外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）中批量分离出8种细胞类型，涵盖髓系细胞、自然杀伤（NK）细胞以及特定的T细胞和B细胞亚群。针对每种批量细胞群体，通过RNA测序（RNA-seq）构建其转录组，共检测到10974个表达基因。对各细胞类型进行两两比较后发现了特异性表达特征；富集分析则在全部8种细胞类型中鉴定出1885至3591个显著富集基因（significantly enriched genes, SEG）。对排名前25%的显著富集基因进行基因本体（Gene Ontology, GO）富集分析，结果显示其显著富集于与各细胞类型特性相关的生物学过程。基因表达比较分析显示，猪细胞与Haemopedia数据库中公开的对应人类PBMC批量RNA测序数据存在极强的相关性。其次，通过对外周血单个核细胞进行单细胞RNA测序（single-cell RNA-sequencing, scRNA-seq），获得了分辨率更高的不同细胞群体转录组数据。共对7份PBMC样本进行分样并测序，最终得到28810个单细胞转录组，这些转录组分布于36个细胞簇中，并被划分为13种通用细胞类型，包括浆细胞样树突状细胞（dendritic cells, DC）、经典树突状细胞、单核细胞、B细胞、经典CD4和CD8 αβ T细胞、NK细胞以及γδ T细胞。基于人类Haemopedia数据库的特征基因集，评估了其在猪细胞表达基因中的相对富集情况；将猪scRNA-seq数据与公开的人类scRNA-seq数据集进行整合，进一步验证了人与猪转录组数据的相似性。分选得到的猪批量RNA测序数据集可为PBMC的scRNA-seq群体分类提供参考；具体而言，数据集整合分析显示，猪批量RNA测序数据有助于明确scRNA-seq数据中的CD4CD8双阳性T细胞亚群。综上，本数据集提供了经充分验证的分选PBMC群体深度转录组数据，同时也是首个猪外周血单个核细胞的单细胞转录组数据集。作为猪动物基因组功能注释（Functional Annotation of Animal Genomes, FAANG）项目的一部分，该资源可为调控免疫遗传性状的猪基因注释提供重要支撑，同时也将为猪免疫学的后续研究及新型试剂开发提供宝贵的数据基础。