FeFo1_Procypris mera
PCR amplification of the V3-V4 region of the 16S rRNA gene was performed using extracted genomic DNA of intestinal microorganisms as a template, with primers 341F: 5'-CCTACGGGGNGGCWGCAG-3' and 806R: 5'-GGACTACHVGGGGTATCTAAT-3'. Three replicates were conducted for each sample, and the PCR products were electrophoresed by 2% agarose gel (Xu et al., 2016). The reaction system (50L) was 5PCR Reaction Buffer10L, 5PCR High GC Enhancer10L, 2.5mM dNTPs1.5L, 10uM Primer F1.5L, 10uM Primer R1.5L, PCR High-Fidelity DNA Polymerase0.2L, 50ng DNA template. The reaction conditions were pre-denaturation of DNA at 95 for 5 min, followed by denaturation at 95 for 1 min, annealing at 60 for 1 min, extension at 72 for 1 min for 30 cycles, and finally extension at 72 for 7 min. The PCR products were detected by color development, a negative control was performed for each detection. Subsequently, the PCR amplification products (~465 bp) were purified and recovered using magnetic beads and quantified by QuantiFluorTM fluorometer. The purified amplification products were uniformly mixed, ligated to sequencing adapters to construct the sequencing library, and then subjected to quality control using a Bioanalyzer and the Agilent High Sensitivity DNA Kit to ensure that the library's peak size was approximately 465 bp. During the base quality check, we used a quality cutoff of 20 and a length cutoff of 45 bp. Reads below these thresholds were removed. After truncation, any reads shorter than 150 bp were discarded to ensure high-quality data. Additionally, we used the Total Count Normalization method, which involves dividing each gene count by the total library size and then multiplying by 1,000,000 to get the number of reads per million (RPM) for each gene. The library was sequenced on the HiSeq platform using the PE250 sequencing technology.