16S rDNA metabarcoding of healthy male sigmoid colon microbiome (H8)
Genomic DNA extraction from the specimens was done using the SkyAmp micro DNA kit (Skygen, Russia) according to the modified protocol. The initial steps of the protocol were modified in the following way. Lysis Matrix Y (MP Biomedicals, USA), and tissue fragments were placed into 1.5 mL Safe-Lock microcentrifuge tubes (Eppendorf, Hamburg, Germany), followed by immediate addition of 180 L of GA Buffer and homogenization for 5 min at 50 Hz in Qiagen TissueLyser LT (Qiagen, Hilden, Germany). Other steps were performed strictly according to the DNA extraction protocol. Particularly, 20 L of Proteinase K was added and vortexed for 10 s. After that, specimens were incubated at 56 C for 30 min in a CH-100 Heating/Cooling Dry Block (BioSan, Latvia). Next, 200 L of GB Buffer and 1 L of the carrier RNA stock solution (1 g/L) were added, and tubes were incubated at 70 C for 10 min. Subsequently, 200 L of ice-cold ethanol was added and tubes were incubated at room temperature for 5 min. The lysates were transferred to the spin columns (in 2 mL collection tubes) and centrifuged for 30 s at 13 400 g. After that the supernatants were discarded, 500 L of GD Buffer was added in every spin column and centrifuged for 30 s at 13 400 g. The last step was repeated twice with 600 L of PV Buffer. Then, the spin columns were centrifuged for 2 min and incubated at room temperature for 5 min. Finally, the spin columns were placed into new 1.5 mL tubes. Twenty-five L of TB Buffer was added in the center of a membrane in every column, and incubated for 5 min at 37 C, followed by centrifuging for 2 min to collect purified DNA. The quality of the extracted DNA was evaluated using 1% agarose gel electrophoresis and a Nanodrop 8000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at OD260/OD280. Qubit 4.0 Fluorometer and dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) were used to measure the DNA concentration. reparation of the DNA libraries was performed according to the Illumina protocol (Part #15044223, Rev. B.) with primers targeting the V3V4 regions of the SSU ribosomal RNA (rRNA) gene, S-D-Bact-0341-b-S-17 (5-CCTACGGGNGGCWGCAG-3) as the forward primer and S-D-Bact-0785-a-A-21 (5-GACTACHVGGGTATCTAATCC-3) as the reverse primer [https://doi.org/10.1093/nar/gks808]. Twenty-five L of the reaction mixture contained both primers (0.4 L each), 1.25 L dNTPs, 0.13 L Q5 High-Fidelity DNA Polymerase (New England Biolabs, USA), 3,75 L Q5 buffer, and 3 L sample DNA.PCR was performed in triplicates for every sample with the following program: 95 C for 3 min, 25 cycles 95 C for 30 s, 56 C for 30 s, 72 C for 30 s, and final extension 72 C for 5 min. The amplicons from triplicates were mixed and cleaned up using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA). The adapters and indices were attached to the amplicons according to the Illumina protocol (Part #15044223, Rev. B). The DNA libraries were validated using real-time PCR, normalized, and pooled. Paired-end 2  250 bp sequencing was performed on the MiSeq platform (Illumina, San Diego, CA, USA) with the Reagent Kit v.2 (Illumina, San Diego, CA, USA).