RNA sequencing analysis of CX3CR1+ cells from Nrp2fl/flLyz2-cre and Nrp2fl/fl mice in colitis model

Nrp2fl/flLyz2-cre and Nrp2fl/fl mice were administered drinking water or 2.5% DSS for 7 days to establish acute experimental colitis model. At day 7, colon tissues from Nrp2fl/flLyz2-cre and Nrp2fl/fl mice (n=3 per group) were isolated and CX3CR1+ cells were enriched with corresponding selection kit following the manufacture’s instruction for RNA sequencing analysis. All the animal experiments were approved by Institutional Animal Care and Use Committee of Sichuan University.UID RNA-seq experiment and high through-put sequencing and data analysis were conducted by Seqhealth Technology Co., LTD (Wuhan, China). Briefly, total RNAs were extracted from samples using TRIzol Reagent. 2 μg total RNAs were used for stranded RNA sequencing library preparation using KC-DigitalTM Stranded mRNA Library Prep Kit for Illumina® following the manufacturer’s instruction. Raw sequencing data was first filtered by Trimmomatic (version 0.36). Low-quality reads were discarded, and the reads contaminated with adaptor sequences were trimmed. Clean Reads were further treated with in-house scripts to eliminate duplication bias introduced in library preparation and sequencing. The de-duplicated consensus sequences were used for standard RNA-seq analysis. Reads mapped to the exon regions of each gene were counted by featureCounts (Subread-1.5.1; Bioconductor) and then RPKM was calculated. Genes differentially expressed between groups were identified using the edgeR package (version 3.12.1). A p-value cutoff of 0.05 and Fold-change cutoff of 2 were used to judge the statistical significance of gene expression differences.