16s rRNA sequencing of uterine flora in infertile human females

DNA Extraction and Amplification: Total genomic DNA was extracted using the MagPure Soil DNA LQ Kit according to the manufacturer's instructions, DNA concentration and integrity were measured using a NanoDrop 2000 and agarose gel electrophoresis, and extracted DNA was stored at -20C until further processing. Extracted DNA was stored at -20C until further processing. The extracted DNA was used as a template for PCR amplification of the bacterial 16S rRNA gene using barcoded primers and Takara Ex Taq. For bacterial diversity analysis, the 16S rRNA gene was amplified using universal primers 343F (5- TACGGRAGGCAGCAG - 3) and 798R (5- AGGGTATCTAATCCT - 3). V3 - V4 (or V4 - V5) variable region of the 16S rRNA gene. Library construction and sequencing: Amplicon quality was visualized by agarose gel electrophoresis, the PCR products were purified by AMPure XP beads and subjected to another round of PCR, and after purification by AMPure XP beads again, the final amplicons were quantified using the Qubit dsDNA Assay Kit and the concentrations were adjusted for sequencing. Sequencing was performed on an Illumina NovaSeq 6000 at 250 bp bipartite.