16S rRNA gene sequencing of fecal samples from mus musculus
Fecal samples were collected from mice in three groups: the necrotizing enterocolitis (NEC) model group, the beta-glucan treatment group, and the healthy control group. Genomic DNA was extracted from the fecal pellets using a commercial DNA extraction kit following the manufacturer's instructions. The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified using specific primers (341F: 5'-CCTACGGGNGGCWGCAG-3' and 805R: 5'-GACTACHVGGGTATCTAATCC-3') with unique barcodes. PCR products were purified using a gel extraction kit and quantified using a Qubit fluorometer (Invitrogen). Sequencing libraries were constructed according to the Illumina Nextera XT Library Preparation Guide. The final libraries were sequenced on an Illumina NovaSeq 6000 platform using a 2300 bp paired-end approach at  Biotree company.