A composite immune signature parallels disease progression across T1D subjects (RNA-Seq Cohort 0 Cell Sorted). A composite immune signature parallels disease progression across T1D subjects (RNA-Seq Cohort 0 Cell Sorted)

Technical replicate testing was performed to determine the measurement precision of each analyte. Peripheral blood mononuculear cells were processed and frozen down from blood samples collected from healthy control subjects (n=3) or subjects with Systemic Lupus Erythematosus (n=3). Three separate PBMC aliquots per subject were then independently thawed and sorted by flow into B cell, CD4+ T cell, CD8+ T cell, and monocyte populations. Total RNA was isolated from the sorted cell populations and then RNAseq libraries were prepared. Overall design: The replicate testing cohort was used to determine the technical precision of each analyte by calculating the intra-subject coefficient of variation (CV) for three replicate aliquots (n=3) per blood draw from 6 subjects. For each analyte, the mean CV across subjects was then calculated. Three independent PBMC aliquots per subject were thawed and sorted by flow cytometry into B cell, CD4+ and CD8+ T cell, and monocyte subsets before RNA sequencing.

本研究通过技术重复检测测定每种分析物的测量精密度。从3名健康对照受试者与3名系统性红斑狼疮（Systemic Lupus Erythematosus, SLE）患者的血液样本中分离得到外周血单个核细胞（peripheral blood mononuclear cell, PBMC）并冻存备用。随后，每名受试者的3份独立PBMC分装样本分别独立解冻，通过流式细胞术分选出B细胞、CD4+ T细胞、CD8+ T细胞与单核细胞群。从分选出的细胞群中提取总RNA，进而构建RNA测序（RNA sequencing, RNA-seq）文库。

实验设计：本重复检测队列通过计算6名受试者单次采血的3份重复分装样本（n=3）的受试者内变异系数（coefficient of variation, CV），测定每种分析物的技术精密度。针对每种分析物，进一步计算所有受试者的平均CV值。每名受试者的3份独立PBMC分装样本经解冻后，通过流式细胞术分选出B细胞、CD4+与CD8+ T细胞及单核细胞亚群，随后进行RNA测序。