Viral RNA Enrichment from Rodent Tissues

The spleen and kidneys of an adult female Rungwe brush-furred rat, snap-trapped in July 2011 in an old banana plantation starting to be overgrown by Afromontane forest on Mount Mabu, Mozambique (coordinates: 16.3086S, 36.4245E), were collected and stored in RNAlater (QIAGEN Benelux, Venlo, The Netherlands). RNA was extracted from one of the kidneys according to the viral enrichment protocol S3, described in [35 (link)], using 25 U RNase ONE Ribonuclease (Promega Benelux, Leiden, The Netherlands) and 30 U Benzonase Nuclease (Sigma-Aldrich, St. Louis, MS, USA) for RNA digestion (90 min at 37 °C). Elution was performed twice by collecting and loading the same eluate on the column. Subsequently, the sample was quantitated using the RNA Quantifluor System (Promega) and an Agilent RNA 6000 Pico chip, loaded on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Viral RNA was subjected to pre-amplification, using the Ovation RNA-Seq System V2 (NuGEN Technologies, San Carlos, CA, USA) for cDNA generation. The sequencing library was constructed with the Ovation UltraLow Library System V2 (NuGEN Technologies) and paired-end sequencing was performed on an Illumina NextSeq 500 (Illumina, Hayward, CA, USA).