Gelred staining

QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) was used to extract genomic DNA according to the manufacturer’s instructions. The extracted DNA was stored at −30 °C until used in the PCR amplification. The presence of E. bieneusi DNA was confirmed by amplifying of an approximately 390 bp fragment including the internal transcribed spacer (ITS) region of the rRNA gene. The primers and PCR thermal cycler parameters used in the present study have been described in our previous study [5 (link)]. Briefly, PCR mixtures (25 μL) were composed of 12.5 μL of Taq mix (Promega, Madison, WI, USA), 1 μL each of the forward and reverse primers (10 μM) (Sunny Biotechnology, Shanghai, China), 1 μL of DNA template and 9.5 μL of nuclease-free water (Promega, Madison, WI, USA). The positive control (E. bieneusi-positive DNA) and negative control (nuclease-free water) samples were added in each PCR run. All the samples were conducted by PCR for three times to make sure the authenticity of the results. The PCR products were examined by 2% agarose gel electrophoresis with GelRed staining (Biotium Inc., Hayward, CA, USA) and observed using a gel imaging system.

Total DNA from MM1 cells was isolated using the QIAmp DNA mini kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. Subsequent bisulfite conversion of 1 µg isolated DNA was performed using the EpiTect Fast Bisulfite Conversion kit (Qiagen, Venlo, Netherlands) and the EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA) for pyrosequencing and Infinium MethylationEPIC analysis, respectively. To confirm successful bisulfite conversion, a PCR using bisulfite-specific primers was executed with the PyroMark PCR kit (Qiagen, Venlo, Netherlands) and the resulting PCR product was visualized on a 2% agarose gel to which GelRed™ staining (Biotium, Fremont, CA, USA) was added. Primers sequences are listed in Supplementary Table S2.

EMSA experiments were done using an 183mer oligonucleotide (Stl binding site₁₈₃) derived from the 171mer oligonucleotide described previously (5 (link)). Stl binding site₁₈₃ (75 ng) and the investigated proteins were mixed in EMSA buffer (PBS (pH 7.3), 5 mM MgCl₂, 75 mM NaCl, 0.5 mM ethylenediaminetetraacetic acid) in the presence or absence of α,β-imido-dUTP (dUPNPP) in 20 μl total volume. Before loading onto 8% polyacrylamide gel the samples were incubated for 15 min at room temperature. Electrophoresis was performed in Tris- Borate- EDTA (TBE) buffer for about 60 min at room temperature, after 1 h pre-electrophoresis. Gels were detected with a Uvi-Tec gel-documentation system (Cleaver Scientific Ltd., Rugby, UK) using GelRed staining (Biotium).

RPRM promoter was amplified from bisulfite-converted DNA using specific primers for methylated and unmethylated DNA, obtained from a previous report (Sato N et al. [8 (link)]). Primer sequences and annealing temperature are provided in Table 1. Bisulfite-modified genomic DNA was amplified by PCR, the cycle of which was: (a) 95 °C for 5 min; (b) 35 cycles: 95 °C 30 s, 59 °C 30 s, and 72 °C 30 s; (c) 72 °C 5 min for a final extension. As a positive control 100 % methylated DNA was used (Zymo Research, Irvine, CA). PCR mix without DNA was used as a blank. PCR products were analyzed by electrophoresis in 1.5 % agarose gel and visualized by gel red staining (Biotium, Hayward, CA) under UV light.

DNA was extracted from an overnight culture of each strain using the wizard genomic DNA purification kit (Promega, Madison, WI, United States) as described by Fernandez et al. (2015) (link). The 16S ribosomal DNA was amplified by PCR (Eppendorf Mastercycler gradient, Hamburg, Germany) with 27f 5′-AGAGTTTGATCMTGGCTCAG-3′ (Gürtler and Stanisich, 1996 (link)) and 1390r 5′-GACGGGCGGTGTGTACAA-3′ (Zheng et al., 1996 (link)) primers (Invitrogen, Carlsbad, CA, United States). The reaction volume was 50 μL, composed of 1 × Taq buffer (New England Biolabs, Beverly, MA, United States), 0.25 U of Taq DNA polymerase (New England Biolabs), 200 nM of each primer, 200 μM of a dNTP mixture (A, T, C, and G, Invitrogen), and 20 ng of bacterial DNA. The thermal cycle program consisted of an initial cycle of 94°C for 5 min for denaturation and polymerase activation, 35 cycles of 94°C for 45 s, 54°C for 45 s, and 68°C for 60 s, and a final extension step of 5 min at 68°C. PCR products were then subjected to gel electrophoresis (100 V, 1 h) on a 1% agarose gel in Tris-acetate-EDTA 1 × buffer (Ambion, Life Technologies Inc., Burlington, ON, Canada) and visualized by gelRed staining (Biotium, Inc., Hayward, CA, United States). Finally, pure PCR products were sequenced using an ABI 3730XL DNA analyzer (Applied Biosystems, Streetsville, ON, Canada).

A nested reverse transcription (RT)-polymerase chain reaction (PCR) assay was used for the detection of enteric viruses. The primers and conditions used in this study are listed in Table 5. For RNA viruses, the cDNA synthesis was performed with the SuperScript^® IV First-Strand Synthesis System (Invitrogen). PCR reactions for EV, AdV, HAV, HEV, RoV, and PMMoV were performed using the MyTaqTM red mix kit (Bioline) in a 25-μL mixture containing 12.5 μL of mix, 1 μL (10 pmol) of each primer, and 2 μL of the extracted genome. In the second amplification cycle, 1 μL of PCR product underwent the nested amplification. Detection of norovirus genogroups I and II (NoV GI and GII) was performed using the PlatinumTM Green Hot Start PCR Master Mix kit (Invitrogen), using the amplification conditions according to the manufacturer’s instructions except for the annealing temperatures, which are reported in Table 5. PCR was performed in a T100 thermal cycler (BioRad, Hercules, CA, USA). Amplicons of the nested PCR were visualized by gel electrophoresis on 2% agarose gels containing GelRed staining (Biotium, CA, USA), then purified using a Montage PCRm96 Microwell Filter Plate (Millipore, MA, USA) and sequenced on both strands by Bio-Fab Research (Rome, Italy). Sequences were compared with available sequences in the GenBank database using BLAST [63 ].