IL-13 promotes sensory-sympathetic neurons crosstalk in asthma [RNA-seq I]

Sequencing of different cell populations of the Jugular-nodose complex (JNC) in a mouse model of Allergic inflammation, and sequencing of cultured JNC nociceptors exposed to IL-13. Overall design: The samples 1A-6A, 1B-6B, 1C-6Ccorrespond to: NaV1.8cre::tdTomatofl/wt mice were injected intranasally with the retrotracer DiD' every day for 3 consecutive days, and underwent (samples 2, 4, 6) or not (samples 1, 3, 5) the OVA model of Allergic Airway Inflammation (AAI). Mice were sacrificed at the peak of inflammation (day 18 of AAI protocol), 15 days after the first tracer injection. For each sample, JNC from 4 mice (2 males and 2 females) were dissected out and pooled together. Ganglia were enzymatically dissociated, nucleus stained with SYTO40, and cells sorted by flow cytometry and collected into Trizol. Airway nociceptors (samples 1A-6A) were gated as Syto40+tdTomato+DiD+, other visceral nociceptors (samples 1B-6B) as Syto40+tdTomato+DiD-, and glial/stromal Nav1.8- cells (samples 1C-6C) as Syto40+tdTomato-. The samples C1-C6 correspond to: NaV1.8cre::tdTomatofl/wt mice were sacrificed and their JNC harvested. For each sample, JNC from 2 males and 2 females were pooled and enzymatically dissociated, seeded at 10000 neurons per well, and cultured with (samples C2, C4, C6) or without (samples C1, C3, C5) 100 ng/mL IL-13 for 24 hours. Cells were then mechanically detached with a cell scraper, sorted by flow cytometry and collected into Trizol. Nociceptors were gated as tdTomato+. RNA from all samples were extracted using the the kit PureLink™ RNA Micro Scale (Thermofisher #12183016), quality controlled, before mRNA library preparation mRNA KAPA mRNA HyperPrep Kit (KapaBiosystems #KR1352). All barcoded samples were then sequenced with a Nextseq500 (Illumina) with 75-cycle single-end read. For analysis, sequences were trimmed for sequencing adapters and low quality 3' bases using Trimmomatic version 0.35 and aligned to the reference mouse genome version GRCm38 (gene annotation from Gencode version M25, based on Ensembl 100) using STAR version 2.7.1. Gene expressions were obtained from STAR as readcounts and computed using RSEM in order to obtain normalized gene and transcript level expression in FPKM.