16S rRNA gene sequencing of colon content samples from mouse treated with iota-carrageenan or kappa-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.

本研究采用十六烷基三甲基溴化铵（CTAB）法，提取小鼠剖检时采集的结肠腔粪便样本中的微生物基因组DNA。通过凝胶电泳对DNA的浓度进行监测并评估其纯度。使用带有条形码（barcodes）标记的特异性引物，对16S核糖体RNA（16S rRNA）基因的高变V3-V4区域进行测序，所用特异性引物信息为：正向引物341F：CCTAYGGGRBGCASCAG；反向引物806R：GGACTACNNGGGTATCTAAT。PCR反应体系采用Phusion®高保真PCR预混液（纽英伦生物技术公司，New England Biolabs）配制。测序文库通过TruSeq® DNA无PCR样本制备试剂盒（Illumina，美国）构建，并在Illumina NovaSeq测序平台上完成测序。