Verity thermocycler

ITS sequences were amplified from extracted DNA using the primer pair UNI_ITS_fw (5′-KRGGRYKAAGTCGTAACAAG-3′) and UNI_ITS_rv (5′-TTTTCRYCTTTCCCTCACGG-3′), targeting the entire spacer region between the 16S rRNA and 23 rRNA genes within the rRNA locus. The amplification was carried out using GoTaq G2 Hot Start polymerase (Promega, USA) on a Verity thermocycler (Applied Biosystems, USA) according to the following protocol: 95°C for 10 min, followed by 32 cycles of 95°C for 1 min, 52°C for 1 min, and 72°C for 1 min and a final step of 72°C for 5 min. The integrity of PCR amplicons was analyzed by gel electrophoresis. The library of ITS amplicons was prepared according to the 16S metagenomic sequencing library preparation protocol (part 15044223, rev. B; Illumina) with modifications in the purification steps. Specifically, the first purification involved 15 μl of Agencourt AMPure XP DNA purification beads (Beckman Coulter Genomics GmbH, Bernried, Germany) in order to remove primer dimers. Then, the second purification step was performed using 30 μl of the above-mentioned purification beads. Sequencing was performed using an Illumina MiSeq sequencer with MiSeq reagent kit v3 chemicals, using 300 cycles.

Fecal samples were collected from WT and KO mice (n = 8–11 for each genotype) receiving either a regular (NC) or a high-fat diet (HFD). All samples were placed immediately into sterile plastic tubes and stored at −80 °C until analysis. DNA was extracted from the samples using the FastDNA SPIN Kit for Soil (MP Biomedicals Inc., Solon, OH, USA) according to the manufacturer’s instructions.
PCR amplification was performed using primers targeting from V3 to V4 regions of the 16S rRNA gene with extracted DNA. The PCR conditions used were 5 min at 95 °C, 35 cycles of 30 s at 94 °C, 30 s at 55 °C and 90 s at 72 °C, followed by 10 min at 72 °C. Amplification was carried out by using a Verity Thermocycler (Applied Biosystems, Forster City, CA, USA). The PCR product was confirmed by using 2% agarose gel electrophoresis and visualized under UV light. The amplified products were purified with the Wizard SV Gen PCR Clean-Up System (Promega, WI, USA). Equal concentrations of purified products were pooled together and followed by a further purification step involving the Agencourt AMPure XP DNA purification beads (Beckman Coulter Genomics GmbH, Bernried, Germany) in order to remove primer dimers. The quality and product size were assessed using a DNA 7500 chip. Mixed amplicons were pooled and the sequencing was carried out according to the manufacturer’s instructions.

Partial 16S rRNA gene sequences were amplified from extracted DNA using primer pair Probio_Uni (5′-CCTACGGGRSGCAGCAG-3′)/Probio_Rev (5′-ATTACCGCGGCTGCT-3′), which targets the V3 region of the 16S rRNA gene sequence^{34 (link)}. Illumina adapter overhang nucleotide sequences were then added to the partial 16S rRNA gene-specific amplicons, which in turn were further processed by employing the 16S Metagenomic Sequencing Library Preparation Protocol (Part #15044223 Rev. B – Illumina; see also below). Amplifications were carried out using a Verity Thermocycler (Applied Biosystems). The integrity of the PCR amplicons was analysed by electrophoresis on a 2200 TapeStation Instrument (Agilent Technologies, USA).