Ileal Microbiota DNA Extraction and 16S rRNA Amplification

According to a suggested method in the previous study, the thawed ileal digesta were digested with 1 mg/mL Protease K (Sigma, St Louis, MO, USA) in a buffer containing 50 mmol/L Tris (pH 7.5), 100 mmol/L EDTA, and 0.5% SDS for 5 h at 55 °C. Ileal microbiota DNA was then isolated by phenol–chloroform extraction following ethanol precipitation [29 (link)]. The concentration of extracted DNA was determined by a Nano-Drop spectrophotometer (Thermo, Wilmington, DE, USA). The V4-V5 region of the bacteria 16S rRNA gene was amplified by polymerase chain reaction (PCR) using primers 515F (5’-barcode- GTGCCAGCMGCCGCGG-3’) and 907R (5’-CCGTCAATTCMTTTRAGTTT-3’), of which the reactions conditions were consistent with previous reports [30 (link)]. PCR products were extracted from 2% (w/v) agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer’s instructions. Then, Purified PCR products were quantified by Qubit®3.0 (Invitrogen, Carlsbad, CA, USA) and used to construct the pair-end library following Illumina’s genomic DNA library preparation procedure [31 (link)]. The raw reads were deposited into the Sequence Read Archive database under the accession number: SRP365994.