Quantum fluorometer

Bacterial DNA was extracted from the fecal samples (0.2–0.3 g) using the E.Z.N.A. soil DNA kit (Omega Bio-tek, Norcross, GA, USA), and the V3–V4 variable regions of bacterial 16S rRNA genes were amplified using an ABI GeneAmp 9700 PCR thermocycler (ABI, Waltham, MA, USA). PCR products were purified using the AxyPrep DNA gel Extraction Kit (AxyPrep Biosciences, Union City, CA, USA). The purified products were quantified with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and a quantum fluorometer (Promega, Madison, WI, USA). Libraries were constructed, then sequenced using the Illumina MiSeq PE300 platform (Illumina, San Diego, CA, USA).

Total DNA from the microbial community in the ruminal fluid from 24 yaks was extracted according to the instructions of the FastDNA^® Spin Kit for Soil (MP Biomedicals, Solon, OH, United States). The determination of DNA concentration and purity, primer selection for PCR amplification, and PCR amplification procedures were performed according to our previous study (Yi et al., 2022 (link)). The PCR products of the same sample were mixed, subjected to 2% agarose gel to recover the PCR products, and extracted using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). A Quantum Fluorometer (Promega, Madison, WI, USA) was used to detect and quantify the recovered products. The sequencing library was constructed and sequenced using the Illumina Miseq technique (Illumina, San Diego, CA, USA) in a manner consistent with our previous study (Yi et al., 2022 (link)). All raw sequences have been uploaded to the NCBI database with the accession number: PRJNA978645.

A quantum fluorometer (Promega, Madison, WI, USA) was utilized to measure the concentration of extracted nucleic acids; preprogrammed settings were available for quantitating DNA (QuantiFluor ds DNA, Promega) and RNA (QuantiFluor RNA, Promega). A volume (100 μL) of diluted nucleic acid sample (1 μL of nucleic acid + 99 μL buffer) was mixed with the appropriate QuantiFluor dye. After 5 minutes of incubation at room temperature, the DNA or RNA concentrations were determined in the fluorometer. The nucleic acid samples were also read in a spectrophotometer, equipped with Nanodrop software (ThermoFisher, Waltham, MA, USA), at 260 nm and 280 nm. If the results were between 1.7 and 1.9, the samples were considered to be contamination free and adequate for further analyses.