RNA sequencing analysis of gene expresssion profiles in spleen and Peyer's patches T follicular helper cells

Purpose: The goal of this study is to compare and examine the transcriptional profiles in spleen(SP) Tfh cells versus Peyer's patches(PP) Tfh cells by mRNA sequencing. Methods: Tfh cells (CD4+CXCR5+PD1+GITR-CD44+) were sorted by a MoFlo-Astrios (Beckman Coulter). Total RNA was extracted using TRIzol Reagent (GeneAll), according to the manufacturer’s instructions. A library for mRNA sequencing was prepared using the SMART-Seq mRNA Kit/NEXtera XT DNA Library Preparation Kit (Illumina) according to the manufacturer’s instructions. Sequencing was performed with Novaseq 6000 (Illumina). Results: For alignment and transcript quantification of raw RNA sequencing data, STAR (v2.7.10b) and RSEM(v1.3.3) were used. To normalize the expected read counts while accounting for differences in sequencing depth and gene length, GeTMM method was applied. Differentially expressed genes (DEGs) analysis was conducted using DESeq2 R package (DESeq2 version 1.42.1; R version 4.1.2) (Log2 fold-change > 1 and P < 0.01). Conclusions: Our study presents comparative gene expression analysis of Tfh cells from SP and PP in wild type SPF mice. We concluded that PP-Tfh cells express higher levels of Tfh related genes. The data reported here also provide supplementary files of read counts and GeTMM