Novaseq 6000 instrument s

Rectal feces were collected from five groups of NOD mice with six samples in each group. DNA was extracted by TIANamp Stool DNA Kit (#DP328‐02; Tiangen) from fecal samples. PCR amplification and library construction were conducted by Phusion enzyme (K1031; APExBIO) and the V3‐V4 region primers (341F 5′‐CCTACGGGNGGCWGCAG‐3′ and 805R 5′‐GACTACHVGGGTATCTAATCC‐3′) of the 16S rRNA gene. An Illumina NovaSeq. 6000 instrument\s was used for paired‐end (PE250) sequencing to collect raw data. Qiime 2 (2020.2) was used to conduct data quality control, calculate the α diversity index, and measure relative abundance. Each amplicon sequence variant/operating taxonomic units (ASV/OTU) sequence was annotated by referring to the SilvA‐132‐99 database, and the corresponding species information and abundance distribution were obtained. R software (Venn Diagram package) and the Jvenn web page (http://www.bioinformatics.com.cn/static/others/jvenn/example.html) were used to analyze common and unique ASVs in groups. Linear discriminant analysis effect size analysis (LefSe, https://github.com/SegataLab/lefse) was applied to evaluate the differential microbiota.

Total RNA was extracted from all aforementioned samples using an RNeasy Plant Mini Kit (QIAGEN) according to the manufacturer’s instructions. The RNA purity and concentration were checked using the NanoDrop 2000 and Qubit 2.0 instruments. Samples with RIN values >8.0 were used for library construction and Illumina sequencing. The libraries were constructed using Illumina TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA, United States) according to the manufacturer’s instructions. Sequencing was performed by Biomarker Ltd. (Beijing, China) on NovaSeq 6000 instruments (Illumina, San Diego, United States) with PE150. To remove low-quality reads or base pairs, reads with adapters, poly-N homopolymers, and very low quality reads were removed from the raw sequencing data. Finally, the Q20 and Q30 values were calculated to further assess the quality of the filtered reads.