DNA extraction and microbiome sequencing

DNA from lung, kidney, ileum, and cecum tissue was isolated (QIAamp DNA stool mini kit; Qiagen, Germantown, MD) following the manufacturer’s protocol with addition of a 10-min incubation at 90°C for better lysis of bacteria. In brief, frozen tissues were weighed and lysed in 1.4 mL of buffer ASL from the QIAamp kit and homogenized with 2.8-mm ceramic beads, followed by 0.5-mm glass beads (OMNI bead ruptor; OMNI International, Kennesaw, GA). Total DNA was quantified (Bioanalyzer 2100 nano chip; Agilent Technologies, Santa Clara, CA). Quantification of bacterial DNA was established by quantitative PCR (Fast SYBR mix, Qiagen) and universal 16S ribosomal RNA (rRNA) bacterial primers UniF1047-1067 (5’-GTGSTGCAYGGYTGTCGTCA) and UniR1174-1194 (ACGTCRTCCMCACCTTCCTC), with nucleotide numbers referring to the location in the 16S rRNA gene of Escherichia coli.²⁸ Cycle parameters were as follows: melting 94°C for 30 s, annealing/extension 60°C for 30 s, for 40 cycles. Standard curves derived from dilutions of bacterial DNA from a 24-h bacterial culture of a fecal sample from a healthy mouse were used to calculate DNA amounts in each sample.¹⁸ Bacterial DNA abundance over time and temperature, as measured by reverse-transcription quantitative PCR (RT-qPCR), were analyzed using 2-way ANOVA with 0 h time point not included in the analysis because it could not be assigned to either the 4°C or to the 20°C groups. However, the 0 h time was included in the analysis of overall bacterial composition.

For sequencing (MiSeq; Illumina, San Diego, CA), total genomic DNA was subjected to PCR amplification targeting the 16S rRNA variable region 4 (V4) using the modified bacterial primers 515F and 806R (http://www.earthmicrobiome.org/emp-standard-protocols/16s/).⁷ Primer sequences without barcode or adapter were forward primer GTGCCAGCMGCCGCGGTAA and reverse primer GGACTACHVGGGTWTCTAAT.⁸ PCR reactions contained PCR-grade water (MilliporeSigma, St. Louis, MO; or MoBio; Qiagen), and master mix (GoTaq green master mix; Promega, Madison, WI), with a final master mix concentration of 1× and final primer concentration of 0.4 µM. Amplification was carried out in 96-well plates at melting 94°C for 45 s, annealing 57°C for 60 s, and extension 72°C for 90 s, for 32 cycles.^7,17

PCR reactions were performed in triplicate for samples of ileum (plus digesta), kidney, and lung tissue, and in duplicate for samples of cecal tissue (and feces). Each set of duplicate and triplicate PCR reactions was pooled for each tissue separately. PCR amplification products were purified for ileum, kidney, and lung samples (MinElute PCR purification kit; Qiagen), and for cecum samples (QIAquick PCR purification kit; Qiagen).

The pools were checked for expected band size of 382 bp (according to reference gene of E. coli)⁸ by gel electrophoresis (2% agarose pre-cast E-gels; Invitrogen, Thermo Fisher Scientific, Waltham, MA) and were visualized under ultraviolet light. Negative controls consisted of water samples without template for DNA extraction and PCR amplification. The pools were then quantified (Qubit dsDNA BR assay kit; Qiagen).

Amplicons were barcoded at the reverse primer with pre-designed 12-bp barcodes as previously described and pooled at equal volumes and concentrations (2 µL of 10 ng/µL DNA).^7,8 The total pool was sequenced (MiSeq; Illumina) at the Center for Genome Research and Biocomputing at Oregon State University (Corvallis, OR) to generate pair-ended 250 nt reads.