Bulk RNA-sequencing of IL-21-tRFP+ CD44+ Th1, Tfh, and memory-like CD4+ T cell subsets on day 14 post LCMV Cl13 infection. Bulk RNA-sequencing of IL-21-tRFP+ CD44+ Th1, Tfh, and memory-like CD4+ T cell subsets on day 14 post LCMV Cl13 infection

CD4+ T cell-derived interleukin 21 (IL-21) sustains CD8+ T cell responses during chronic viral infection, but the helper subset that confers this protection remains unclear. Here, we applied scRNA and ATAC-seq approaches to determine the heterogeneity of IL-21+CD4+ T cells during LCMV clone 13 infection. Overall design: IL-21-tRFP+ CD44+ CXCR6+ (Th1), CXCR5+ (Tfh), and CXCR6-CXCR5- (memory-like) CD4+ T cells were FACS-sorted from LCMV Cl13-infected IL-21-tRFP 10Bit reporter mice on day 14 p.i. ( 5,000 cells per subset from 3 replicate (independent) donor mice were used to prepare RNA-seq libraries, which were prepared using SMART-seq technology (Picelli et al., 2014) and sequenced on an Illumina NextSeq.

CD4+ T细胞来源的白细胞介素21（interleukin 21, IL-21）在慢性病毒感染过程中可维持CD8+ T细胞应答，但介导该保护效应的辅助T细胞亚群仍未明确。本研究采用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）与转座酶可及性染色质测序（assay for transposase-accessible chromatin sequencing, ATAC-seq）技术，解析淋巴细胞脉络丛脑膜炎病毒克隆13（lymphocytic choriomeningitis virus clone 13, LCMV clone 13）感染过程中IL-21+CD4+ T细胞的异质性。

总体实验设计：于感染后第14天，从感染LCMV Cl13的IL-21-tRFP 10Bit报告小鼠中，通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分选出IL-21-tRFP+ CD44+ CXCR6+（1型辅助T细胞，Th1）、CXCR5+（滤泡辅助T细胞，Tfh）以及CXCR6-CXCR5-（记忆样）CD4+ T细胞亚群；每组从3只独立供体小鼠中获取约5000个细胞，采用SMART-seq技术（Picelli等，2014）构建RNA测序文库，并在Illumina NextSeq测序平台完成测序。