16S Seq of gut microbiota: adult female gut (HFpEF)
Fecal samples were obtained from the National Medical Research Center for Therapy and Preventive Medicine of the Ministry of Healthcare of the Russian Federation. Human stool samples were donated by patients and healthy volunteers who signed informed consent in accordance with the local Ethic Committee (protocol No. 04-05/18 date: 7 June 2018, protocol No. 03-02/19 date: 11 April 2019). Immediately after delivery, the stool samples were frozen at 80 C until use. After thawing, the stool aliquots were separated into groups for two independent sets of DNA extraction. Each aliquot was obtained by taking a 200 mg scraping from each individual fecal sample. DNA was extracted using either QIAamp Fast DNA Stool Mini (Qiagen, Germany) or QIAamp PowerFecal Kit (Qiagen, Germany) according to the manufacturers instructions. A dsDNA HS Assay Kit and Qubit 4.0 fluorometer (Thermo Fisher Scientific, the USA) were used to measure the DNA concentration, and the quality of isolated DNA was analyzed by electrophoresis in 1% agarose gel. DNA libraries were prepared using polymerase chain reaction (PCR) amplification with gene-specific primers for the V3V4 regions of 16S rRNA. Processes, such as DNA fragmentation, repair, adapter ligation, and unbiased PCR amplification, were performed strictly in accordance with Illumina instructions. The quality of the prepared libraries was analyzed on the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) using the High Sensitive DNA kit (Agilent Technologies, USA). A Qubit dsDNA HS Assay Kit and Qubit 4.0 fluorometer were used to measure the DNA concentration in each sample. The quality-qualified libraries were pooled in a 0.2-mL PCR tube. A MiSeq Reagent Kit V2 Nano was used to prepare the DNA for sequencing. Finally, the samples were sequenced using the Illumina MiSeq platform (Illumina, San Diego, CA, USA) according to the manufacturers protocol using reagents for double-ended reading and a read length of at least 250 b.p. The amount of PhiX Control v3 was not less than 1%.