Colibri microvolume spectrometer

RNA was quantified using a Colibri® microvolume spectrometer (TITERTEK BERTHOLD) and normalized for cDNA synthesis. cDNA synthesis was performed using a Verso cDNA Synthesis Kit (Thermo Scientific™, Cat# AB-1453/A). Quantitative real-time PCR (qRT-PCR) was carried out using synthesized cDNA as a template and a 2X DyNAmo ColorFlash SYBR Green qPCR Kit (Thermo Scientific™) in Realplex4 epgradient Mastercycler (Eppendorf). Primers used for qRT-PCR analysis are listed in Table S1. Relative quantification of gene expression was done via the ^ΔΔCt method. The 16 s rRNA gene was used as the housekeeping gene in our study. Log₂ fold change cutoff ≥1.5 was considered to indicate upregulation, and ≤ −1.5 was considered to indicate downregulation.

DNA extraction. Herpesvirus-DNA was extracted from 400 μl of bile and 200 μl of serum using QIAamp DNA blood mini-kit (Qiagen, Germany). To extract DNA from the formalin-fixed paraffin-embedded liver biopsies, the QIAamp DNA FFPE tissue kit (Qiagen, Germany) was used. Both serum and bile samples were stored at − 80 °C, and extracted DNA was temporarily stored at − 20 °C. The DNA concentration after extraction was measured with a Colibri microvolume spectrometer (Titertek-Berthold, Germany).
Quantitative real time polymerase chain reaction (qPCR). Virus-specific r-gene quantification kits (Biomerieux, France) were used to prepare the extracted DNA for quantification. The kit contains an internal control that was added to the samples before DNA extraction to monitor the extraction process and the presence of amplification inhibitors. Appropriate negative controls were performed to check for contamination along the extraction and amplification process. Quantification standards were available for HHV 1–6 allowing the measurement of the viral DNA load. The detection of HHV-7 was qualitative, and the HHV-7 viral count could not be quantified. PCR was performed using the LightCycler 480 (Roche Diagnostics, Switzerland).

A total of 10 fecal samples were collected directly from the rectums of the five obese dogs at the 0-week and 10-week time point. By using the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany), the total DNA was extracted from a total of 200 mg of each dog fecal sample. With the 2 cycles using taco™ Prep Bead Beater (GeneReach, Taichung, Taiwan), the total DNA from fecal samples were extracted by bead-beating, including heating in a water bath (5 min incubation at 70 °C between beatings). For measuring the concentrations of DNA, the Colibri Microvolume Spectrometer (Titertek Berthold, Pforzheim, Germany) was used with OD260/280 ratios of 1.80–2.15 in each DNA sample.

Total RNA was isolated from snap frozen liver samples (RNeasy Plus Mini Kit, Qiagen, Germany). The quantity of RNA was measured on Colibri Microvolume Spectrometer (Titertek-Berthold, Germany). 500 ng of total RNA was used to be reversely transcribed into cDNA using High-Capacity cDNA Reverse Transcriptase Kit (ThermoFisher, Germany) according to the manufacturer’s instructions. Each qRT-PCR reaction was performed using 2 µl of cDNA in a final volume of 10 µl. All samples were run in duplicates. RT-PCR was performed using the following TaqMan Gene Expression Assays: il-1β Mm00434228, tnf-α Mm00443258, ifn-γ Mm01168134, il-12a Mm00434169, il-12b Mm00434174_m1, il-4 Mm00445259, acta-2 Mm00725412, il-13 Mm00434204, il-10 Mm01288386 (ThermoFisher, Germany). Cycling was performed using QuantStudio 3 (Thermo Fisher Scientific, Germany) under the following reaction conditions: 50°C for 2 min followed by 95°C for 10 min, 45 cycles at 95°C for 15 s, and at 60°C for 1 min. The ΔΔCt method was utilized for relative quantification (16 (link)). Gene expression values were normalized to the endogenous reference gene GAPDH (Rodent GAPDH control reagent, ThermoFisher, Germany) and presented as normalized expression values relative to naive controls.

Strains were grown on nutrient agar (Merck, Darmstadt, Germany) in petri dishes for 24 h at 37 °C, and the biomass was harvested and used for subsequent DNA isolation. For short-read sequencing, DNA was isolated using the Genomic-tip 100/G kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. For long-read sequencing, DNA was extracted using the NucleoBond HMW DNA kit (Macherey-Nagel, Düren, Germany). The genomic DNA content and purity were checked by Qubit 3.0 Fluorometer (QubitTM DNA HS Assay, Life Technologies, Thermo Fisher Scientific Inc., Eugene, OR, USA) and Colibri Microvolume Spectrometer (LB 915, Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany) measurement. Sequencing library preparation was performed using the Nextera XT library preparation kit (Illumina Inc., San Diego, CA, USA) for Illumina sequencing and the Ligation Sequencing Kit SQK-LSK 109 in combination with the Barcoding Kit EXP-NBD 104 (Oxford Nanopore Technologies Ltd., Oxford, England) for ONT, both according to the manufacturer’s recommendations. The Illumina libraries were sequenced on a MiSeq system (Illumina) producing paired-end reads. For long-read sequencing, the libraries were run on a MinION Mk1B sequencing device (Oxford Nanopore Technologies Ltd.) for 24 h using a R9.4.1 flow cell.