title: Virome of Non-human primates:feces
design: These non-human primates included rhesus macaques, pigtailed macaques, sooty mangabey, and chimpanzee, and captive in three zoos in eastern China. Fecal 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 ĄÁ g) and filtered through a 0.45-_m 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 37ĄăC for 90 min . Viral nucleic acids protected from digestion within viral capsids and other small particles were then extracted using QiaAmp Mini Viral RNA kit (Qiagen) according to the manufacturerĄŻs instructions. Seven separate pools of nucleic acids from 69 fecal specimens were randomly generated, 6 of which contained 10 fecal specimens and the other one contained 9 fecal specimens. These seven 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