Miseq v3 chemistry

DNA was extracted from each frozen brain sample. The entire mtDNA was amplified with two overlapping long-range PCR amplicons (Primers sequences in Supplementary Table 1, online resource), tagged and indexed applying Illumina Nextera XT reagents and sequenced at high depth with Illumina Miseq v3 chemistry. Each DNA sample was sequenced twice, including independent PCR amplification, library preparation and sequencing.

All S. sonnei confirmed as CipR.MSM5 isolated at Sciensano’s reference laboratory between January 2017 and March 2022 and with sequence data were included. All (n = 112) isolates came from patients aged up to 84 years who were mostly male (n = 101/112, 90%). Sequence data was obtained using the following protocol: isolates were cultured overnight in BHI broth (BD) at 37 °C. DNA was extracted using an MgC Bacterial DNA Kit™ with a 60 μL elution volume (Atrida, Amersfoort, The Netherlands), following the manufacturer’s instructions. Sequencing libraries were constructed using the Illumina Nextera XT DNA sample preparation kit and sequenced on an Illumina MiSeq instrument with a 250 bp paired‐end protocol (MiSeq v3 chemistry), according to the manufacturer’s instructions (Illumina, San Diego, CA, USA).

ChIP was performed as described previously^{72 (link)} with some modifications. In brief, HA-tagged MafB overexpressed MS1 ECs were cross-linked with 1% (wt/vol) formaldehyde for 10 min at room temperature and then added glycine to a final concentration of 0.15 M. Chromatin was sonicated to an average fragment length of 150–250 bp using SFX250 digital sonifier (Branson) for 2 min: 0.7 s on/1.3 s off on ice and immunoprecipitated with anti-HA antibody-conjugated magnetic beads (M180-11; MBL) overnight at 4 °C. Following protein–DNA complex elution, samples were treated with RNaseA and proteinase K and incubated at 65 °C overnight for reverse cross-linking. Immunoprecipitated DNA was purified using AMPure XP SPRI beads (Beckman Coulter). Sequencing libraries were prepared with NEBNext Ultra DNA Library Prep Kit for Illumina (New England BioLabs) according to the manufacturer’s instructions and sequenced on a MiSeq sequencer using 2 × 75 bp paired-end MiSeq v3 chemistry (Illumina). Sequencing reads were aligned to the mm10 reference mouse genome using Bowtie2^{73 (link)} and analyzed with HOMER^{31 (link)} for peak finding, motif analysis and peaks annotation. Gene set annotation enrichment tests were performed using GREAT v.3.0^{32 (link)} with default parameters and were subsequently used for calculating the overlap with DEGs in the Mafb-cKO RNA-Seq experiment.

16S rRNA sequencing analysis was done in the Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, and referred to Nakayama’s protocol [20 (link)]. The stool genomic DNA was amplified using TaKaRa ExTaq HS (Takara Bio, Shiga, Japan), targeting the V3-V4 region (F (Bakt_341F): CGCTCTTCCGATCTCTGCCTACGGGNGGGWGCAG, R (Bakt_805R): TGCTCTTCCGATCTGACGACTACHVGGGTATCTAATCC). The obtained amplicons were then used as a template for secondary PCR, which amplification was with barcode-tag primers. Furthermore, the secondary PCR product of 28 samples was mixed and subjected to paired-end sequencing using an Illumina MiSeq v3 chemistry (Illumina inc, San Diego, CA, USA).

DNA of the sediment samples was extracted from 0.5 g of sediment using the Power Soil DNA Isolation Kit (MO BIO Laboratories; Carlsbad, CA, United States), following the manufacturer’s instructions. The extracted DNA samples were quantified and sent for sequencing at Genoinseq (Cantanhede, Portugal) company. The hypervariable V4-V5 regions of the 16S rRNA gene were amplified using the universal primers 515YF (5-GTGYCAGCMGCCGCGGTAA-3) and Y926R (5-CCGYCAATTYMTTTRAGTTT-3), developed by Parada et al. (2016) (link). Pair-end sequencing with MiSeq® V3 chemistry was performed according to the manufacturer’s instructions (Illumina, San Diego, CA, United States). Raw reads extracted from Illumina MiSeq ® System were demultiplexed and pre-processed. Sequences were quality-filtered with PRINSEQ software and merged by using the AdapterRemoval v2.1.5 software (Schubert et al., 2016 (link)) with default parameters. Full description of the protocol is reported in (Bragança et al., 2019 (link)). Raw Illumina fastq files obtained in this study were deposited in the European Nucleotide Archive (ENA) database under the accession number PRJEB59507.

DNA was extracted using a modified protocol of the Qiagen DNeasy plant kit (80 (link)), quantified using the QuBit double-stranded DNA (dsDNA) high-sensitivity assay (Life Technologies, Grand Island, NY). For amplicon sequencing, DNA was diluted with Tris-EDTA (TE; pH 8) to 1 ng · μL⁻¹, with V1-V2 16S rRNA amplicon sequencing and PCRs performed as described previously (34 (link)). Each reaction mixture included 5 ng template, 5 μL 10× buffer, 1 U Hi-Fi Taq, 1.6 μL 50 mM MgSO₄ (Life Technologies), and 200 nM (each) forward primer 27F_ill 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGagrgttygatymtggctcag‐3′ and reverse primer 338RPL_ill 5′‐GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGgcwgccwcccgtaggwgt‐3′; capital letters represent Illumina linker sequences on the 27F/338R primer pair (81 (link)). Reactions were cycled for 2 min at 94°C, 15 sec at 94°C (30 times), 30 sec at 55°C, 1 min at 68°C, and 7 min 68°C (for elongation). Purification was done using the MinElute kit (Qiagen, Valencia, CA), and product presence and removal of primer-dimers were verified by electrophoresis. V9 18S rRNA amplicons were generated as described above using the 1389F/1510R primer pair (24 (link)). Samples were sequenced using Illumina MiSeq v3 chemistry.

DNA was quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). 250 ng DNA was sheared in the Covaris® S2 instrument (Covaris, Inc.) to an insert size of approximately 650 bp. Fifty nanograms of sheared DNA was used for preparation of sequencing libraries with the ThruPLEX® DNA-seq Kit (Rubicon Genomics) according to the manufacturer’s instructions using provided primers with ten cycles for amplification. Primer sequences were as follows: 5’: AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT with NNNNNNNN being a TruSeq HT i5 index and 3’: GTTCGTCTTCTGCCGTATGCTCTANNNNNNNNCACTGACCTCAAGTCTGCACACGAGAAGGCTAGA with NNNNNNNN being a TruSeq HT i7 index.
PCR products were purified on the MBS Magnatrix 1200 automated workstation (NorDiag) with Dynabeads® MyOne carboxylic acid beads (Thermo Fisher Scientific). Purity of the samples and insert size distribution were inspected using the High Sensitivity DNA Kit on the Agilent 2100 Bioanalyzer instrument (Agilent Technologies). DNA libraries were quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and equimolar concentrations of 12 samples were pooled and further purified using Agencourt AMPure XP (Beckman Coulter, Inc.). Library pools with a final concentration of 10 nM DNA were submitted to one flow cell per pool and sequenced using the MiSeq V3 chemistry (Illumina).