Dynamic alterations of the mice gut virome after Coxsackievirus B3 infection:mousefe016
For viral metagenomic analysis, eight male BALB/c mice (4 to 8 weeks) were injected intraperitoneally with Coxsackievirus B3 or phosphate-buffered saline (PBS) as mock infection (n=3). Mice were individually housed in separate cages within a specific pathogen-free barrier facility and feces were collected before infection and on the 3rd, 5th and 7th day after infection. 20 samples from 5 mice in Coxsackievirus-infected group and 12 samples from 3 mice in healthy control group were collected and stored in ultra-low temperature refrigerator. Each sample was added 1mL Dulbeccos phosphate-cushioned saline (DPBS) and vortexed for 5min tremendously. The supernatants were collected after centrifugation (5 min, 15,000g). 500 uL of each supernatant was filtered through a 0.45 micron filter (Millipore) to remove eukaryotic and bacterial cell-sized particles. 200 uL of supernatant from each samples was then treated with a mixture of nuclease enzymes to digest unprotected nucleic acids. The remaining total nucleic acids were isolated using a QIAamp Viral RNA Mini Kit (QIAGEN) according to the protocol. Extractions were treated with a reverse transcription kit (SuperScript III Reverse Transcriptase) with six-base random primers to reverse transcribe RNA into cDNA. The Klenow fragment polymerase (New England Biolabs) was then added to synthesize the second strand of cDNA (dsDNA). The resulting dsDNA products were used to construct 32 libraries, using a Nextera XT DNA Sample Preparation Kit (Illumina) and then sequenced using an Illumina NovaSeq platform with 250bp paired ends with dual barcoding for each individual sample pool.