Hiseq 2500 instrument

For RNA extraction, 50 ml ABG aliquots were inoculated from starter cultures (5 ml LB broth) with an OD₆₀₀ of 0.03 and incubated at 37 °C with agitation (220 rpm). Synthetic valdiazen resuspended in methanol was added from the start to a final concentration of 50 µM when appropriate. An equal volume of methanol was added to all cultures not containing synthetic valdiazen. The final methanol concentration in all cultures was 0.1%. Three independent cultures were grown to an OD₆₀₀ of 0.9 to 1 and harvested, using 1/10 of stop solution (10% phenol buffered with 10 mM Tris-HCl pH 8). RNA extraction and genomic DNA removal were performed as previously described^{58 (link)}. A total of 150 ng good quality RNA (RNA integrity factor > 6) were further processed (cDNA synthesis and library preparation) using the Ovation® Complete Prokaryotic RNA-Seq Library System from NuGEN. After quantification of the obtained cDNA libraries^{59 (link)}, Illumina single-end sequencing was performed on a HiSeq2500 Instrument. The CLC Genomics Workbench v7.0 (QIAGEN CLC bio, Aarhus, Denmark) was used to map the sequencing reads to the B. cenocepacia H111 reference sequence^{60 (link)}. Statistics and differential analysis was done using the DESeq software^{61 (link)}

RNA was sent to the Tufts University Core Facility for library preparation and sequencing using the Illumina TruSeq RNA library preparation kit and the HiSeq 2500 instrument. Sequencing data were analyzed using the Tuxedo Suite as previously described (31 (link)). NCBI genome Candida albicans SC5314 (assembly ASM18296v3; accession no. GCF_000182965.3) and annotation GFF file were used as the reference genome. A bowtie2 index was created using bowtie2-build (v2.2.1) from the fna sequence file, while the downloaded GFF was converted to GTF using gffread for downstream use. The raw sequencing reads for each sample were mapped to the bowtie2 index using bowtie2 (v2.2.1) with default parameters and then sorted and converted to bam format using samtools v1.9 (sort function). The resulting sorted bam files were used as input for Cuffdiff (Cufflinks v2.1.1) with the triplicates grouped. Genes with a fold change of over 2 were considered.

Purified RNA from the HDMVEC is submitted to the University of California, San Diego (UCSD) Institute for Genomic Medicine core facility for library preparation and high-throughput next-generation sequencing. Libraries are constructed using TruSeq Stranded mRNA Library PrepKits (Illumina, San Diego, CA) and run on a HiSeq. 2500 instrument (Illumina). Sequence reads are quantitated using Whippet (version 1.6.1) (76 (link)) with the GRCh38.p13 RefSeq genome annotations. Differential expression analysis and PCA analysis are performed with DESeq2 (version 1.34.0) (77 (link)). KEGG pathway analysis is performed with significant differentially expressed genes (adjusted P-value < 0.05) using clusterProfiler (version 4.2.2) (78 –80 (link, link)).

DNA was extracted from fecal samples with the QIAamp fast DNA stool minikit (Qiagen, Hilden, Germany). Shotgun metagenomic sequencing was performed on all samples with an Illumina HiSeq 2500 instrument. Libraries were constructed using NEBNext Ultra DNA library prep kit for Illumina (NEB, USA) following the manufacturer’s recommendations to generate DNA fragments of ∼300 bp; paired-end reads were generated by sequencing 150 bp in the forward and reverse directions.
A total of 93 stool samples were shotgun sequenced (n of 17 and 14 for Probio-M8 and placebo groups; sampling at days 0, 30, and 90 for each individual), generating 630.62 Gbp of high-quality paired-end reads (6.78 ± 0.97 Gbp/sample; range = 4.45 to 9.69 Gbp) for downstream analysis. Low-quality (sequences shorter than 60 nucleotides [nt]) and host-contaminated reads were filtered through the KneadData quality control pipeline (http://huttenhower.sph.harvard.edu/kneaddata).

DNA was extracted from fecal samples using methods described in the Human Microbiome Project [62 (link)]. In brief, microbial cells in fecal homogenates were physically disrupted by bead-beating, and then DNA was purified using a Mo Bio PowerSoil DNA isolation kit. The DNA was checked for quantity and quality using a combination of gel electrophoresis, nanodrop spectrometry, and Qubit fluorometry. The DNA was shotgun sequenced by NZ Genomics Ltd using Nextera library preparation and pools of 12 barcoded samples run per lane on an Illumina HiSeq 2500 instrument (Illumina).
Gut microbiome sequence data were analyzed using recognized computation (bioinformatics) tools [27 (link)]. These tools include the preparation of species-sampling curves that are the classic means of evaluating ecological richness (alpha diversity—biodiversity). As the genomes of all microbial species present in the microbiota were sequenced as small DNA fragments, both phylogenetic (describing what kinds of microbes are there and their relative abundances; MetaPhlAn2, QIIME v2) and functional (the biochemical capacity encoded in the metagenome; HUMAnN2) information are available [61 ].

PicoPure RNA Isolation Kit (Applied Biosystems™, Vilnius, Lithuania) was used to extract the total RNA from isolated LE, GE, BV, and stromal cells of individual pig following the manufacturers’ instructions. After RNA isolation, each RNA sample was performed on the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) with the Agilent RNA 6000 Pico assay to assess RNA integrity and quantity. RNA Integrity number (RIN) of all samples ranged from 6.1 to 8.7, and most samples’ RIN number were around 7.5. Total RNA with 800 pg input was used for starting the library preparation, then a number of 32 RNA samples with 4 biological replicates in each cell type were prepared following the Ovation SoLo Single Cell RNA-Seq System (NuGen Technologies, San Carlos, USA). It was worth to notice that the number of PCR cycles was set with 16 during the amplification. Finally, a total number of 32 individual libraries with unique barcodes were mixed within three pools for one lane sequencing with single-read flow cell on an Illumina HiSeq 2500 instrument. The process of sequencing and demultiplexing was provided by the Functional Genomics Center Zurich (FGCZ).