Precellys evolution instrument

Fecal samples were briefly centrifuged, ethanol removed, and the pellet used for DNA extractions with the DNAeasy Powersoil kit (Qiagen), following the manufacturer’s protocol. Samples were homogenized with 0.1 mm glass beads at 5,500 rpm, 2 ×45 s using a Precellys Evolution instrument (Bertin Technologies). DNA quality was assessed with Nanodrop and sent to the Centre for Genomic Regulation (CRG) in Barcelona (Spain) for amplicon generation and sequencing. The region V3-V4 of 16S rRNA was amplified using a pool of five forward and reverse primers (including a frameshift to increase diversity) with Nextera overhangs (Table S2). For each sample, amplicons were generated in three-replicates using KAPA Hifi DNA polymerase (Roche), with a first round of PCR (25 cycles); amplicons were then pooled and a 5 µl purified aliquot was used to seed the second PCR (8 cycles) for individual barcoding. Two negative controls (water only) and two mock communities (HM-277D and HM-276D from BEI Resources) were processed along with sample DNA. Barcoded amplicons were pooled at equimolar concentrations and the final library cleaned with the Sequal kit (Invitrogen). The library was sequenced on Illumina MiSeq v3 (600-cycle cartridge, 300 paired-end reads). The final sample dataset did not include any recaptured specimens.

IL-1β, IL-6 and TNFα were quantified from full ear skin samples (dermis + epidermis) following allergen re-exposure. The skin was removed from the cartilage tissue using surgical tweezers, and snap-frozen in liquid nitrogen 24 h post challenge. Samples were submerged in 800 µL lysis buffer (50 mM Trizma base, 250 mM NaCl, 6.4 mM EDTA, 17.6 mM Triton X-100, pH 7.4, including protease inhibitor cocktail (cOmplete, Roche Diagnostics GmbH, Berlin, Germany)). Tissue homogenization was performed in MK28 hard grinding precellys tubes (Bertin Technologies, Montigny-le-bretonneux, France) using a Precellys Evolution instrument (Bertin Technologies). Protein concentration in the supernatant was quantified by a Bradford assay and stored at −80 °C. The protein concentration of all samples was adjusted to 2 mg/mL. IL-1β levels were analyzed using a mouse IL-1β/IL-1F2 DuoSet ELISA kit (R&D systems), on 10× diluted samples. IL-6 and TNFα levels were analyzed using respective ELISA MAX™ standard kits (Biolegend) on undiluted samples. All assays were performed following manufacturer’s protocol.

Harvested pellets were resuspended in 500 μl 2× Tris-EDTA (TE) buffer with 1.2% Triton X-100. Cells were lysed using lysing matrix B tubes (MP Biomedicals) and a Precellys Evolution instrument (Bertin Instruments) at 7,400 rpm for 2 rounds of 40 s, and genomic DNA was isolated using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s instructions with the following modifications: twice the amount of supernatant, proteinase K, buffer AL, and ethanol were used prior to adding samples to each column.