scRNAseq gene expression and VDJ-TCR analysis of PBMCs from patients with tattoo uveitis and sarcoidosis

To elucidate the complexity of immune responses in the formation of granuloma, we performed the single-cell RNA-sequencing analysis. PBMCs derived from tattoo uveitis patients in both active and inactive stages and sarcoidosis patients were collected to characterize their similarities and difference. PBMCs from age-matched healthy donors were considered as controls. Overall design: Sorted live PBMCs were prepared two patients with active tattoo uveitis, three patients with convalescent tattoo uveitis, three patients with sarcoidosis and three age-match healthy donors. Single-cell gene expression and TCR VDJ libraries were prepared by Chromium Next GEM Single Cell 5' Kit v2 Dual Index and Single Cell Human TCR Amplification Kit (10x Genomics) with the help from Biomolecular Resource Facility at ANU. Libraries were then sequenced on NovaSeq 6000 sequencer (Illumina) with 2 x 50 bp paired-end reads. 10X Cell Ranger (10X Genomics, v6.0.1) was utilized to align the reads to the human genome (GRCh38-2020-A) using default parameters.

为阐明肉芽肿形成过程中免疫应答的复杂性，我们开展了单细胞RNA测序分析。收集了活动期与非活动期纹身性葡萄膜炎患者、结节病患者的外周血单个核细胞（Peripheral Blood Mononuclear Cells, PBMCs），以解析其异同特征。以年龄匹配的健康供者的外周血单个核细胞作为对照。

总体实验设计：分选获得的活体外周血单个核细胞分别来自2例活动性纹身性葡萄膜炎患者、3例恢复期纹身性葡萄膜炎患者、3例结节病患者及3例年龄匹配的健康供者。本研究借助澳大利亚国立大学（Australian National University, ANU）生物分子资源中心的技术支持，采用10x Genomics的Chromium Next GEM单细胞5'试剂盒v2双索引组合与单细胞人T细胞受体扩增试剂盒，完成单细胞基因表达文库与T细胞受体VDJ文库的构建。随后在Illumina NovaSeq 6000测序仪上以2×50 bp双端读长模式进行测序。使用10X Cell Ranger（10x Genomics, v6.0.1），以默认参数将测序读段比对至人类参考基因组GRCh38-2020-A。