Picogreen dsdna assay kit

The OMEGA Soil DNA Kit (M5635‐02) (Omega Bio‐Tek) was used to extract the genomic DNA of stool samples, the DNA was quantified by Nanodrop, and the quality of DNA extraction was detected by 1.2% agarose gel electrophoresis. Based on the 16S rRNA V3–V4 region of microorganisms, the corresponding primers were designed: F: ACTCCTACGGGAGGCAGCA; R: GGACTACHVGGGTWTCTAAT, and the sample‐specific Barcode sequence was added, and then the rRNA gene V3–V4 fragment was amplified by PCR. The amplification products were purified and recovered by magnetic beads, and the PCR amplification products were quantified by fluorescence. The fluorescent reagent was Quant‐iT PicoGreen dsDNA Assay Kit, and the quantitative instrument was Microplate reader (BioTek, FLx800). According to the fluorescence quantitative results, each sample is mixed in a corresponding proportion according to the sequencing volume requirement of each sample. Illumina NovaSeq sequencing was used to prepare the sequencing library using Illumina's TruSeq Nano DNA LT Library Prep Kit.

The total sample DNA was used as the template for the PCR amplification of the bacterial 16S rRNA gene in the V3 + V4 region. The V3 + V4 variable region common primer was synthesized by Shanghai Personal Biotechnology Co., Ltd. The primer sequence was 338F: 5′-ACTCCTACGGGAGGCAGCA-3′ and 806R: 5′-GGACTACHVGGGTWTCTAAT-3′. PCR amplification system: 25 μL: 5 × reaction buffer 5 μL, 5 × GC buffer 5 μL, dNTP (2.5 mM) 2 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, DNA template 2 μL, ddH₂O 8.75 μL, and Q5 DNA polymerase 0.25 μL. Amplification parameters: initial denaturation at 98 °C for two minutes, denaturation at 98 °C for 15 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, and final extension at 72 °C for 5 min; 29 cycles. The PCR products were detected by agarose gel electrophoresis and purified using the agarose gel recovery kit (Shao et al. 2020 (link)). The fluorescence reagent used was the quant-it PicoGreen dsDNA Assay Kit, and the quantitative instrument performed using a microplate reader (BioTek, FLx800).
The sequencing library was prepared according to the instructions of the TruSeq Nano DNA LT Library Prep Kit (Illumina). The library concentration was > 2 nM. The amplified V3 + V4 variable region of the 16S rRNA was subsequently sequenced using the Illumina MiSeq platform (Frasergen Co., Ltd.).

Libraries were prepared using the Tecan Ovation SoLo RNA-Seq library preparation kit as per the manufacturer’s instructions. Libraries were quantified using the Quant-iT PicoGreen dsDNA assay kit and read on a BioTek FLx800 fluorescence microplate reader. Libraries were pooled equal molar and the sample pool was quantified using Biorad ddPCR. Libraries were sequenced as stated below.