Metagenomic of Cynopterus bat rectal swab sample (treated sample)
Two aliquots of sample in question were retrieved from the -80 C freezer and centrifuged at 1800 x g and 4C, for 15 min. Eukaryotic and bacterial cell debris was removed by passing the supernatants through a filter with 0.45 m pores. The first aliquot (the untreated sample) was used for nucleic acid extracted with the RNA Easy Fast Tissue/Cell kit (TIANGEN). In the second aliquot (treated sample), the unprotected nucleic acids were digested with a cocktail of enzymes  15 U Turbo DNase, 20 U Benzonase, 20 U RNase One, in 10x Turbo DNase buffer  and 30 mM EDTA was then added and incubated with the mixture for 30 minutes to stop the enzymatic reaction (120,121). The nucleic acids obtained from the two sample aliquots were then subjected to reverse transcription with the ProtoScript II First-Strand cDNA Synthesis Kit (New England Biolabs), followed by second-strand synthesis with the NEBNext Ultra II Non-Directional RNA Second-Strand Synthesis Module (New England Biolabs). Two libraries were constructed with the NEBNext Ultra II library preparation kit, in accordance with the protocol recommended by the manufacturer (Illumina) for use with FFPE RNA. The libraries were pair end-sequenced on an Illumina Novogene 6000 device.