Total RNA was purified from mLN Tn sorted from 4-week- (Monarch Total RNA Miniprep Kit, NEB, Ipswich, Massachusetts/US) or 3-month-old rats (RNeasy Micro Kit, QIAGEN, Hilden - Germany). Samples with RNA integrity number > 7 were sequenced. Libraries were prepared using NEBNext Low Input RNA Library Prep Kit (3-month-old) or NEBNext rRNA Depletion Kit and NEBNext Ultra II Directional RN Library Prep Kit (4-week-old), and sequenced on an Illumina HiSeq 2000 (3-month-old) or NextSeq 500/550 (4-week-old), paired-end 75 bases. Sequencing was performed at the genomic platforms of Cochin Institute—Paris Centre University, or Faculty of Health Simone Veil–Versailles-Saint-Quentin/Paris-Saclay University. Volcano plots were designed with EnhancedVolcano. Transcripts were aligned to the rat genome Rnr_6.0 using STAR version 2.7.1 then reads were counted using featureCount (14 (link), 15 (link)). Differential analysis was performed with DESeq2 including batch effect (16 (link)). Pathways analysis was performed using QIAGEN Ingenuity Pathway Analysis (IPA) (17 (link), 18 (link)). Upstream transcription regulators were retrieved using IPA, computing an overlap p-value (overlap between the dataset genes and the genes regulated by a transcription regulator) and an activation z-score, which infers the predicted activation state of the transcription regulator.
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