Biodrop duo

Nasal swabs were placed in 500 μL de PBS, vortex for 30 s and stored at −80 °C until used for DNA extraction. DNA was extracted from 200 μL of the initial 500 μL PBS where the swabs were resuspended and eluted in 100 μL of PBS using the Nucleospin Blood (Macherey Nagel, Bethlehem, PA, USA) kit. Quantity and quality assessment of the DNA was performed using BioDrop DUO (BioDrop Ltd., Cambridge, UK). Samples were submitted for 16S rRNA gene amplicon sequencing using the Illumina paired-end 2 × 250 bp kit (MS-102-2003 MiSeq^® Reagent Kit v2, 500 cycle) following the manufacturer’s instructions. The library preparation for sequencing was performed within 24 h after the DNA extraction at Servei de Genòmica, Universitat Autònoma de Barcelona. The region amplified was V3–V4 that covers two hypervariable regions of the conserved gene, and it was sequenced according to the Illumina protocol [27 (link)] as previously described [6 (link)]. The entire sequence dataset is available at the NCBI database, SRA accession number PRJNA717778.

Total RNA from cells was extracted with TRIzol reagent (Life Technologies, Grand Island, NY, USA) following the protocol provided by the manufacturer. RNA concentration was measured using BioDrop Duo (Biodrop, Cambridge, UK). cDNA was synthesized from 2 µg of total RNA using the High-Capacity cDNA Reverse Transcription System (Life Technologies). Comparative quantitative RT-PCR (qPCR) was performed in duplicate for each sample using SYBR^® Premix Ex Taq^TM (Life Technologies) and CFX CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). RT-PCRs were performed using the primers listed in Table 1. Expression levels of mRNA were quantified by use of the threshold cycle (Ct) method. Ct values for each gene of in­terest were normalized to GAPDH.

Total RNA was isolated using TRIzol reagent (Life Technologies, Grand Island, NY, USA) following the protocol provided by the manufacturer [68 (link),69 (link)]. RNA concentration was measured using BioDrop Duo (BioDrop, Cambridge, UK), and cDNA was synthesized by using a High-Capacity cDNA Reverse Transcription System (Life Technologies). qPCR was performed in duplicate for each sample using SYBR^® Premix Ex Taq^TM (Life Technologies) and a CFX CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). qPCR was performed using the primers listed in Table 1. The mRNA expression level of genes of interest was normalized to that of GAPDH.

Cells were seeded on Petri dishes (60 mm) and incubated in standard conditions until 90% confluence was achieved. Then, cells were treated with experimental medium as described in Section 5.2. After 24 h, total RNA was isolated using TRIzol reagent, according to the manufacturer’s instruction. BioDrop DUO was used to determine RNA purity and concentration (Biodrop, Cambridge, UK). 5 µg of RNA from each sample was used to synthesize cDNA by using ImProm RT-IITM reverse transcriptase (Promega, Madison, WI, USA). LightCycler 96 (Roche, Basel, Switzerland) was used to perform the RT-qPCR with 2 µL of cDNA. Primer-BLAST was used to design primers. To calibrate reaction, Human Reference RNA (Stratagene, San Diego, CA, USA) was used. Ribosomal protein S17 (RPS17), ribosomal protein P0 (RPLP0), and histone H3.3A (H3F3A) were used as a reference genes. Sequences of primers used in the study are presented in Table 5. Specificity of received product was confirmed during analysis of melting curves for each reactions. The ΔΔCt method was used to analyze the obtained data. The experiment was performed in duplicate with three independent replications.

Total DNA was extracted from 300 mg of resuspended feces in 900 µl of PBS using the Nucleospin Blood kit (Machinery Nagel). Purified DNA was eluted in a final volume of 50 μl of elution buffer (5 mM Tris, pH 8.5). The quality and quantity of genomic DNA was evaluated on a BioDrop DUO (BioDrop Ltd). The library preparation for sequencing was performed within 24 h after the DNA extraction, at Servei de Genomica, Autonomous University of Barcelona.

Nasal swabs were taken from piglets belonging to each group at different timepoints (D0, D7, D15, D21, D49) for bacterial culture, PCR or microbiota analysis, as described in Table 2.
Nasal swabs were resuspended in 500 µL of PBS and kept refrigerated until arrival at research facilities where they were vortexed and stored at − 20 °C. A total of 200 µL of the suspensions was processed using the Nucleospin Blood kit (Macherey–Nagel, Düren, Germany) according to the manufacturer’s instructions. Total extracted DNA was quantified using BioDrop DUO (BioDrop Ltd., Cambridge, UK) and stored at − 20 °C for further processing.

Total RNA was isolated using TRIzol reagent (Life Technologies, Grand Island, NY, USA) following the protocol provided by the manufacturer. RNA concentration was measured using BioDrop Duo (Biodrop, Cambridge, UK), and cDNA was synthesized by a High-Capacity cDNA Reverse Transcription System (Life Technologies). qPCR was performed in duplicate for each sample using SYBR^® Premix Ex Taq^TM (Life Technologies) and CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). qPCR was performed using the primers listed in Table 1. Expression levels of mRNA were normalized to GAPDH.