Inulin_16S rRNA seq_2
DNA extraction was performed using repeated bead beading protocol which was developed by Yu and Morrison (29) and described in detail by Koc et al (27). Pellets were resuspended using 1 mL of lysis buffer composed of 500 mM NaCl, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA, and 4% sodium dodecyl sulfate (SDS). The resulting suspension was transferred to 2 mL screw-cap microtubes (Sarstedt, Nmbrecht, Germany) containing Zirconia/Silica beads (0.125 g each of 0.1 mm and 1.5 mm beads, along with a single 2.5 mm bead; Biospec, Oklahoma, USA). The mixture was subjected to mechanical disruption using a bead beater (Biospec, Oklahoma, USA) at 4000 rpm for 3 minutes. Following this, the tubes were incubated at 70C for 15 minutes. The samples were then centrifuged at 16,000 g for 5 minutes at 4C. The supernatant was carefully transferred to a sterile 2 mL Eppendorf tube, while 300 L of the lysis buffer was added to the pellet, and the homogenization, incubation, and centrifugation steps were repeated. The collected supernatants were combined in a 2 mL tube. Next, 260 L of chilled 7.5 M ammonium acetate was added to the supernatant, and the mixture was incubated on ice for 5 minutes. The tubes were centrifuged again at 16,000 g for 10 minutes at 4C, after which 650 L of the supernatant was transferred into two separate 1.5 mL tubes. An equal volume (650 L) of isopropanol was added to each tube, and the samples were incubated overnight at -20C. After incubation, the samples were centrifuged at 16,000 g for 15 minutes at 4C. The supernatant was discarded, and the pellet was washed with 200 L of 70% ethanol. The pellet was then resuspended in 100 L of Tris-EDTA buffer. To degrade RNA, 2 L of RNase (10 mg/mL) was added, followed by incubation at 37C for 15 minutes. Afterward, 15 L of proteinase K (from the QIAamp DNA Stool Mini Kit) and 200 L of AL buffer (from the same kit) were added to the samples, which were incubated at 70C for 10 minutes. Subsequently, 200 L of absolute ethanol was added to precipitate the DNA. The samples were then transferred to QIAamp mini columns, and the wash and elution steps were carried out according to the QIAamp kit protocol. Finally, the extracted DNA was stored at -20C until it was needed for 16S rRNA library preparation.DNA samples were processed following the Illumina 16S Metagenomics Sequencing Library Preparation protocol. Amplification targeted the V3-V4 regions of the 16S rRNA gene using 16S Amplicon PCR Forward Primer = 5 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and 16S Amplicon PCR Reverse Primer = 5 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATC. PCR reactions were carried out on a 2720 thermal cycler (Life Technologies) using KAPA HiFi HotStart ReadyMix. The PCR conditions consisted of an initial denaturation at 95C for 5 minutes, followed by 25 cycles of 95C for 30 seconds, 55C for 30 seconds, and 72C for 30 seconds, with a final elongation step at 72C for 5 minutes. For indexing, the Illumina Index primer set was applied following the manufacturers instructions. The resulting PCR amplicons were visualized on a 1.5% agarose gel (Sigma-Merck, Germany) stained with SYBR Safe (Thermo Fisher, MA, USA) to confirm amplification. Subsequently, PCR products were purified using the AMPure XP bead system (Beckman Coulter, CA, USA) in accordance with the Illumina protocol. Prior to pooling, the concentration of the purified products was quantified using the Qubit dsDNA High-Sensitivity kit (Thermo Fisher, MA, USA) and the Qubit 3.0 Fluorometer (Thermo Fisher, MA, USA). Following library preparation, all subsequent libraries were precisely pooled to achieve a final concentration of 4 nanomolar (nM). Afterwards, sequencing was performed utilizing the NextSeq 2000 platform, leveraging the high-throughput capabilities of this technology. Specifically, sequencing utilized the P1 600-cycle (2x300 bp) reagent kit sourced from Illumina (San Diego, USA). The sequencing procedures were conducted at the Teagasc Moorepark Research Centre Next Generation DNA Sequencing Facility.