THE FECAL VIROME OF CATS IN AN ANIMAL SHELTER
These cats had been in the shelter between a few days up to 2 weeks. They were kept in separate cages in a single large room, where the cages were stacked 3 high 10 across. The cats were fed the same food which changed depending on the donations received. Stool samples were resuspended in 10 volumes of phosphate-buffered saline (PBS) and vigorously vortexed for 5 min. Four hundred microliters of supernatant was collected after centrifugation (10 min, 15,000 × g) and filtered through a 0.45-µm filter (Millipore) to remove eukaryotic and bacterial cell-sized particles. The filtrates enriched in viral particles were treated with a mixture of DNases (Turbo DNase from Ambion, Baseline-ZERO from Epicentre, and benzonase from Novagen) and RNase (Fermentas) to digest unprotected nucleic acid at 37°C for 90 min . Viral nucleic acids protected from digestion within viral capsids and other small particles were then extracted using magnetic beads of MagMAX Viral RNA Isolation kit (Ambion) according to the manufacturer’s instructions. Six separate pools of nucleic acids from 25 fecal specimens were randomly generated, 5 of which contained 4 fecal specimens and the other one contained 5 fecal specimens. These six viral nucleic acid pools, containing both DNA and RNA viral sequences, were then subjected to reverse transcription reactions with SuperScript III reverse transcriptase (Invitrogen) and 100 picomoles of a random hexamer primer, followed by a single round of DNA synthesis using Klenow fragment polymerase (New England BioLabs). A library was then constructed using Nextera XT DNA Sample Preparation Kit (Illumina) and then sequenced using the Miseq Illumina platform with 250 bases paired ends with a distinct molecular tag for each pool