16S rRNA Gene Amplification and Sequencing

Following manufacturer instructions of the PF Mag-Bind Stool DNA Kit (Omega Bio-tek, Norcross, GA, USA), we extracted microbial genomic DNA. PCR targeted the 16S rRNA gene V3–V4 region using 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) primers under the following conditions: initial denaturation at 95 °C for 3 min, followed by 27 cycles of denaturing at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 45 s, and single extension at 72 °C for 10 min, and end at 4 °C [27 (link)]. Each sample had 3 replicates, pooled and purified from 2% agarose gel, and size-verified. Quantification was carried out using Promega’s Quantus™ Fluorometer (Promega, Madison, WI, USA). Purified PCR products underwent Bioo Scientific’s NEXTFLEX Rapid DNA-Seq Kit library prep (Austin, TX, USA). Illumina’s PE300/PE250 platforms (Illumina, San Diego, CA, USA) were sequenced by Shanghai Meiji Biomedical Technology Co., Ltd. (Shanghai, China) Raw data were submitted to the NCBI SRA database.