Rna-seq of bat, Rhinolophus affinis: viral rna pool of pharyngeal and anal swab specimens collected from bats in Tunchang, China
Swab samples were thawed and vortexed for resuspension in maintenance medium, then subjected to filtration via a 0.45 m membrane filter (Millipore Sigma, Burlington, MA, USA) to eliminate cellular debris. Following ultracentrifugation at 100,000g for 3 hours at 4C, the resulting pellets were resuspended in Hanks' Balanced Salt Solution. Enzymatic digestion was carried out to eliminate residual nucleic acids using a cocktail of TURBO DNase (Ambion), benzonase (Novagen), and RNase One (Promega). Viral DNA and RNA were concurrently isolated using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). First-strand cDNA was synthesized and subsequently converted into double-stranded form using Klenow fragment (NEB, Ipswich, MA, USA). A sequence-independent PCR was conducted, and the amplified products were purified using a MinElute PCR Purification Kit (Qiagen) in preparation for downstream analyses.