Enbnext ultra directional rna library prep kit

Total RNA was extracted from mouse lungs using the TRIzol^® reagent (Thermo Fisher Scientific) according to the instructions of the manufacturer (n = 4–5, each group). The concentration and purity of the RNA samples were determined by automated optical density evaluation (OD₂₆₀/OD₂₈₀ ≥ 1.8 and OD₂₆₀/OD₂₃₀ ≥ 1.8) using a NanoDrop spectrophotometer (Thermo Fisher Scientific). RNA sequencing (RNA-seq) libraries were prepared using a NEBNext rRNA Depletion Kit (New England Biolabs, Ipswich, MA, USA) and an ENBNext Ultra Directional RNA Library Prep Kit (New England Biolabs) according to the instructions of the manufacturer using 500 ng of the total RNA samples. Next, 2 × 36 base paired-end sequencing was performed using a NextSeq 500 sequencer (Illumina, San Diego, CA, USA) by Tsukuba i-Laboratory LLP (Tsukuba, Japan). Sequences were mapped to the mm10 mouse genome and quantified using CLC Genomics Workbench version 10.1.1 (QIAGEN). An adjusted P-value <0.01 (Benjamini–Hochberg FDR method for multiple testing corrections) and relative changes in transcription levels >1.2-fold were used as the cutoff criterion (Supplemental Figure 1). The data are available under GEO series accession number GSE181966.

The RNA-Seq method was described previously in detail^{54 (link)}. Briefly, total RNA was extracted from spleens and inguinal lymph nodes using TRIzol reagent according to the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, MA). The RNA-Seq library was prepared from 50 ng of RNA by using the ENBNext Ultra Directional RNA Library Prep Kit (New England Biolabs (NEB), Ipswich, MA) after the depletion of rRNA (NEB NEBNext rRNA Depletion Kit). Paired-end sequencing (2 × 36 bases) was carried out by using NextSeq.500 (Illumina, San Diego, CA). Sequence reads were mapped to the mouse genome (mm10) using CLC Genomics Workbench (Version 7.5.1; Qiagen, Redwood City, CA). The expression level of each gene was estimated as ‘normalization values’ by CLC Genomics Workbench or CLC Main Workbench^{55 (link)}. Differential expression was analysed by empirical analysis using the Empirical Analysis of DGE tool (edgeR test) in CLC Main Workbench.