16S rRNA gene sequencing of colon content samples from mouse treated with or without κ-carrageenan

The fecal samples collected from the colon lumen of mice at necropsy were used to extract microbial genomic DNA using CTAB method. Gel electrophoresis was used to monitor the concentration and evaluate the purity of DNA. The hypervariable V3-V4 regions of the 16S rRNA gene were sequenced with specific primers (forward primer, 341F: CCTAYGGGRBGCASCAG; reverse primer, 806R: GGACTACNNGGGTATCTAAT) tagged with barcodes. The PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs). The sequencing library was generated using TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) and was sequenced on an Illumina NovaSeq platform.