Qiaamp fast dna stool mini kit

Total DNA from the fecal microbiota was extracted using the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions (QIAamp Fast DNA Stool Mini Kit Handbook, www.qiagen.com/handbooks). The degree of degradation and potential contamination of DNA was analyzed by electrophoresis using 1% agarose gel. DNA purity was determined using a NanoPhotometer^® spectrophotometer (IMPLEN, Westlake Village, CA, USA), and DNA concentration was measured using the Qubit^® double-stranded DNA (dsDNA) Assay Kit on a Qubit^® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). One microgram of the qualified DNA was used to construct the library. DNA samples were fragmented to 350 base pairs (bp) by sonication, and the DNA fragments were end-polished, A-tailed, and ligated with a full-length adaptor for Illumina sequencing with further polymerase chain reaction (PCR) amplification. The libraries were analyzed for size distribution using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and quantified via real-time PCR. Libraries were sequenced using the Illumina PE150 platform (Illumina, Inc., San Diego, CA, USA).

BALF precipitation samples (1-3mg) were weighed in 2 ml microcentrifuge tubes and placed on ice. Total DNA from the lower respiratory tract microbiotas was extracted using the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Germany) per the manufacturer’s instructions (see the QIAamp Fast DNA Stool Mini Kit Handbook, www.qiagen.com/handbooks). The degradation degree and potential contamination of the DNA were analyzed using 1% agarose gels. The DNA purity was determined using the NanoPhotometer^® spectrophotometer (IMPLEN, CA, USA). DNA samples were further diluted with sterile water to an OD value between 1.8 and 2.0, measuring with the Qubit^® dsDNA Assay Kit in Qubit^® 2.0 Fluorometer (Life Technologies, CA, USA). One microgram of qualified DNA was used to construct the library via NEBNext^® Ultra DNA Library Prep Kit for Illumina (NEB, USA). DNA samples were fragmented to 350 bp by sonication, and then the DNA fragments were end-polished, A-tailed, and ligated with the full-length adaptor for Illumina sequencing with further PCR amplification. Libraries were analyzed for size distribution using the Agilent2100 Bioanalyzer (Agilent, USA) and quantified via real-time PCR to keep size distribution of DNA fragments >3nM. The libraries were then sequenced on an Illumina PE150 HiSeq platform.

DNA extraction was performed using a QIAampTM Fast DNA Stool Mini Kit (Qiagene, No. 51604). High-throughput sequencing of 16S rDNA gene amplicons was performed by Novogene Biotech Co., Ltd. (Beijing, China) using a NovaSeq PE250 platform (Novogene Biotech Co., Ltd, Beijing, China). The high-quality sequences were clustered into operational taxonomic units (OTUs) at a 97% similarity level, and a total of 1808 OTUs were obtained. And then, the OTUs sequences were annotated with Silva132 database. According to the species annotation, the α diversity and β diversity were further calculated, and the differences between groups were compared to reveal the different characteristics of microbial community structure under different treatments.

DNA was extracted from 180–220 mg of the cecal samples using a QIAampTM Fast DNA Stool Mini Kit (Qiagene, No. 51604) according to the manufacturer’s instructions. Total DNA was quantified using a Thermo NanoDrop 2000 UV microscope spectrophotometer and 1% agarose gel electrophoresis. 16S rDNA high-throughput sequencing was performed by Realbio Genomics Institute (Shanghai, China) using the Illumina Hiseq PE250 platform. The V3-V4 region of the 16S rDNA gene was amplified using the universal primers, 341F (CCTACGGGRSGCAGCAG) and 806R (GGACTACVVGGGTATCTAATC). The raw pair-end reads were merged and quality-filtered to remove tags with lengths < 220 nt, an average quality score of <20, and tags containing >3 ambiguous bases using PANDAseq (v2.9) [17 (link)]. Singletons and chimeras were removed, and the resulting quality-filtered sequences were clustered into 97% operational taxonomic units (OTUs) using USEARCH (v7.0.1090) in QIIME software. The Ribosomal Database Project (RDP) algorithm trained on the Greengenes database was used to classify each OTU (http://greengenes.lbl.gov). The open source software package QIIME (http://qiime.org) was used to measure alpha diversity (including the chao1, observed species and PD whole tree indices). The metagenomic reads have been submitted to the NCBI-SRA database under accession number SRP159441.

The replicates of 16S sequencing are n = 6 for CT and XOS group and n = 7 for BBC and MIX group. The extraction of bacterial DNA was conducted as described by our previous study (Wu et al., 2018 (link)). Briefly, DNA was extracted from 180–220 mg of the cecal samples using a QIAampTM Fast DNA Stool Mini Kit (Qiagen, cat# 51604) according to the manufacturer’s instructions. Total DNA was quantified using a Thermo NanoDrop 2000 UV microscope spectrophotometer and 1% agarose gel electrophoresis. The 16S rDNA high-throughput sequencing was conducted by Realbio Genomics Institute (Shanghai, China) through an Illumina Hiseq PE250 platform. The universal primers 341F (CCTACGG GRSGCAGCAG) and 806R (GGACTACVVGGGTATCTAATC) were used to amplify the V3-V4 region of the 16S rDNA gene. The raw pair-end reads were merged and quality-filtered by using PANDAseq (v2.9) to remove tags with lengths <220 nt, an average quality score of <20, and tags containing >3 ambiguous bases (Masella et al., 2012 (link)). The USEARCH (v7.0.1090) in QIIME software was applied to cluster the quality-filtered sequences into 97% operational taxonomic units (OTUs). Each OTU was classified by the Ribosomal Database Project (RDP) algorithm trained on the Greengenes database.⁴ The alpha diversity was determined by the software package QIIME.⁵