Agencourt ampure xp dna purification bead

The DNA library constructions derived from PCR were purified twice by magnetic separation using Agencourt AMPure XP DNA purification beads (Beckman Coulter Genomics). The elimination of free primers and new concentrations of libraries were then verified with electrophoresis on an Agilent 2100 Bioanalyser (Agilent Technology). All amplicons were diluted to 26 pM—except for controls (dissection [1 sample], extraction [3 samples], and PCR [2 samples]), which were used pure—and pooled to equalize concentrations for emulsion PCR with the Ion One Touch 400 Template kit v2 (Life Technologies) according to the manufacturer’s instructions. Sequencing of the amplicon libraries was carried using the Ion PGM™ Hi-Q™ Sequencing kit with 48 barcoded amplicon libraries pooled on Ion 318™ chips.

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.

PCR products obtained following the amplification of a section of the 16S rRNA gene were purified by a magnetic purification step involving Agencourt AMPure XP DNA purification beads (Beckman Coulter Genomics GmbH, Bernried, Germany) in order to remove primer dimers. The DNA concentration of the amplified sequence library was estimated employing a fluorimetric Qubit quantification system (Life Technologies). Amplicons were diluted to 4 nM and 5 µl of each diluted DNA amplicon sample was mixed to prepare the pooled final library. Paired-end sequencing (250 bp × 2) was performed using an Illumina MiSeq sequencer with MiSeq Reagent Kit v3 chemicals-600 cycles (Illumina Inc., California, USA).

PCR products obtained following amplification of part of the 16 S rRNA gene sequences were purified by a magnetic purification step involving Agencourt AMPure XP DNA purification beads (Beckman Coulter Genomics GmbH, Bernried, Germany) in order to remove primer dimers. The DNA concentration of the amplified sequence library was estimated through fluorimetric Qubit quantification system (Life Technologies). Amplicons were diluted to 4 nM and 5 μl of each diluted DNA amplicon sample was mixed to prepare the pooled final library. Sequencing was performed using an Illumina MiSeq sequencer with MiSeq Reagent Kit v3 chemicals.

Partial 16S rRNA gene sequences were amplified from extracted DNA using the primer pair Probio_Uni (5′-CCTACGGGRSGCAGCAG-3′) and Probio_Rev (5′-ATTACCGCGGCTGCT-3′) targeting the V3 region of the 16S rRNA gene sequence (37 (link)). Illumina adapter overhang nucleotide sequences were added to the partial 16S rRNA gene-specific amplicons, which were further processed by means of the 16S metagenomic sequencing library preparation protocol (part 15044223, rev. B; Illumina). Amplifications were carried out using a Verity thermocycler (Applied Biosystems). The integrity of the PCR amplicons was analyzed by electrophoresis on a 2200 TapeStation instrument (Agilent Technologies, USA). DNA products obtained following PCR-mediated amplification of the 16S rRNA gene sequences were purified by a magnetic purification step employing Agencourt AMPure XP DNA purification beads (Beckman Coulter Genomics GmbH, Bernried, Germany) in order to remove primer dimers. DNA concentration of the amplified sequence library was determined by a fluorometric Qubit quantification system (Life Technologies, USA). Amplicons were diluted to a concentration of 4 nM, and 5-μl quantities of each diluted DNA amplicon sample were mixed to prepare the pooled final library. Sequencing was performed using an Illumina MiSeq sequencer with MiSeq reagent kit v3 chemicals.

The archaeal 16S, eukaryotic 18S and fungal ITS1 genes were PCR amplified using primers listed in Supplementary Table 1. The resulting PCR products were purified using Agencourt AMPure XP DNA purification Bead (Beckman Coulter, USA) and quantified using Nanodrop-1000 (Thermo Scientific, USA). Then, PCR products of all NGTs samples (n = 19), all New-DMs (n = 14) and all Known-DMs (n = 16) were pooled by mixing equal quantities of concentration normalized PCR products. This way we obtained three pools for each group, NGTs, New-DMs and Known-DMs for archaeal 16S rRNA, eukaryotic 18S rRNA and fungal ITS1 genes. All the pooled samples were then sequenced using Ion Torrent PGM. Since fungal ITS amplicons varied in length, we fragmented 100 ng of it with Ion Shear Enzyme mix (Ion Xpress Plus Fragment Library preparation kit, Life Technologies) for 20 min and 200 bp size fragments were selected before adapter ligation step (Tang et al., 2015 (link)).

The functional frc-gene was PCR amplified using primers frc171-F (5′-CTSTAYTTCACSATGCTSAAC-3′) and frc306-R (5′-GDSAAGCCCATVCGRTC-3′) as described earlier15 (link). The resulting PCR products were purified using Agencourt AMPure XP DNA purification Bead (Beckman Coulter, USA) and quantified using Nanodrop-1000 (Thermo Scientific, USA). Then, PCR products were pooled by mixing equal quantities of concentration normalized PCR products in five group pools. All the pooled group samples were then sequenced as described above.