RNA-seq and 16S rRNA-seq for b3gnt8 KO and Wt mice

Emerging evidence suggests that alterations in intestinal epithelial glycosylation are implicated in the pathogenesis of inflammatory bowel disease (IBD). However, the intricate roles of gut glycosylation in maintaining intestinal homeostasis remain inadequately elucidated. Beta 1, 3-N-acetylglucosaminyltransferases (B3GNTs) are Golgi glycosyltransferases involved in the biosynthesis of poly-N-acetyl-lactosamine chains. In this study, we here create <i>B3gnt8</i> knockout (<i>B3gnt8</i>^{<em>−/−</em>}) mice to investigate its precise effects on intestinal homeostasis.RNA-Seq (RNA Sequencing) was conducted by Sangon Biotech (Shanghai, China). Total RNA was extracted from the small intestinal and colonic mucosa of <i>B3gnt8</i>^<em>-/-</em> mice (n = 3-5) and <i>Wt</i> mice (n = 3 - 5), including samples from the proximal small intestine, distal small intestine, and colon; each group had five biological replicates. Total microbial genomic DNA was extracted from feces of <i>B3gnt8</i>^<em>-/-</em>(n = 5) and <i>Wt </i>mice (n = 5) using a DNeasy PowerSoil Kit (QIAGEN, Venlo, Netherlands) following the manufacturer's instructions. PCR amplification of the bacterial 16S rRNA gene V4–V5 region was conducted with forward primer 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and reverse primer 907R (5′-CCGTCAATTCMTTTRAGTTT-3′), incorporating sample-specific 7-bp barcodes for multiplex sequencing. PCR amplicons were purified with Agencourt AMPure Beads (Beckman Coulter, Indianapolis, IN, USA) and quantified using a PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA).