RNA sequencing analysis of LPS-treated BMDMs from Nrp2fl/flLyz2-cre and Nrp2fl/fl mice

Primary bone marrow-derived macrophages (BMDMs) were isolated from femurs of Nrp2fl/flLyz2-cre and Nrp2fl/fl mice and cultured in DMEM supplemented with 10% FBS, penicillin-streptomycin (50 U/mL) and 20% L929 supernatant as a source of M-CSF for 7 days maturation. After 7 days, BMDMs were treated with 200 ng/ml LPS for 18 h in vitro. After polarization, BMDMs were collected 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.