Stool dna extraction kit

Cecal contents were collected from some animals and frozen at − 80 °C. DNA was extracted using Stool DNA Extraction Kit (Qiagen) according to the manufacturer’s instructions. To check DNA quality and 16S content prior to sequencing, universal primers were used for SYBR Green quantitative polymerase chain reaction (qPCR) with the following extended cycling protocol: 95 °C 10 min; 95 °C 15 s, 60 °C 30 s, 72 °C 30 s for 40 cycles. Sequencing was completed at the Cincinnati University Children’s Hospital Medical Center’s DNA Sequencing and Genotyping Facility Core (Cincinnati, OH) as described [14 (link)]. All antibiotic-treated samples failed to yield 16S rDNA sequence data; one sample each from the ethanol- and pair-fed groups was excluded based on insufficient sequence data.
UPARSE and UTAX (http://www.drive5.com/usearch/manual/cmd_utax.html) were used to generate OTU tables from 16S rDNA read data and to make taxonomic assignments. QIIME package scripts were used for calculations of α- (PD_whole_tree, chao1, observed_otus and shannon) and β- (Bray-Curtis, Un-Weighted UniFrac, and Weighted UniFrac) diversity.

Genomic DNA of the stool samples was extracted utilizing the Stool DNA Extraction Kit (Qiagen, Germany), and the extracted DNA was purified employing a DNA gel purification kit (Qiagen, Germany). The quality of the extracted DNA was assessed by 1.0% agarose gel and stored at −80°C for sequencing.
The specific primers 338F and 806R were used to amplify the hypervariable region V3–V4 of the 16S rRNA gene (Meng et al., 2020 (link)). PCRs were run in 20.0-μl reaction mixtures containing 4 μl 5 × FastPfu Buffer, 2 μl 2.5 mM dNTPs, 0.8 μl forward primer (5 μM), 0.8 μl reverse primer (5 μM), 0.4 μl FastPfu Polymerase, 0.2 μl BSA, 10 μl template DNA, and 1.8 μl ddH₂O, in an ABI GeneAmp 9700 instrument (ABI, United States). The thermal cycling program was as follows: 95°C for 3 min, 30 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 45 s, and finally, 72°C for 10 min. The total purified PCR products with a concentration >10 ng/μl and an OD260/OD280 ≈ 1.8 were subjected to sequencing on an Illumina MiSeq platform at Shanghai Meiji Biomedical Technology (Shanghai, China). The sequence data were submitted to the NCBI Sequence Read Archive with accession PRJNA721594.¹

DNA from stool samples was extracted using a Qiagen Stool DNA extraction kit and processed as previously described [40 (link)]. The extracted stool DNA was investigated for 16S rRNA gene-based bacterial diversity using a Human Intestinal Tract microarray (HITChip) as previously described [40 (link)]. The tissue samples (cancers and matched normal) were first lysed for homogenization. Then, 3 cycles of freeze-thawing were performed followed by bead-beating to achieve bacterial cell disruption. DNA from the resulting lysates was extracted using the Qiagen AllPrepDNA/RNA kit (Qiagen, Inc., Germantown, MD, USA).