Truseq rna library preparation kit

Library preparation, sequencing and metagenomics analysis of the pooled R. tanezumi tissues were performed as previously described [17 (link)]. Briefly, the metagenomic library was prepared with the TruSeq RNA library preparation kit (Illumina, San Diego, USA) and sequenced on an Illumina MiSeq platform with 150 bp single-end reads. The subsequent FASTQ file was trimmed and taxonomically classified using DIAMOND v0.9 [18 (link)]. Jeilongvirus reads were extracted and mapped to a closely related genome sequence (MT085297) identified through BLASTn, with a full-length genome obtained and annotated for reference. This dataset is available at the NCBI Sequence Read Archive under BioProject accession number PRJNA990547. For whole genome sequencing of individual small mammal samples (n = 3), RNA was treated with the RiboZero Plus rRNA depletion kit (Illumina, San Diego, USA), prior to library preparation with the TruSeq RNA library preparation kit (Illumina, San Diego, USA) following the manufacturer’s instructions. The library was quality checked and sequenced on an Illumina MiSeq platform with 250 bp paired-end reads. FASTQ files were quality checked and trimmed with BBDuk2, before mapping to a reference genome created from the metagenomic dataset (PRJNA990547) to obtain the full-length genomes.

RNA extraction and transcriptome sequencing were carried out as described by Padovan et al. [27 (link)]. Briefly, the samples were ground to a fine powder in a mortar and pestle under liquid nitrogen. Total RNA was extracted using the Spectrum Total RNA Kit as per the manufacturer’s instructions (Sigma Aldrich, MO, USA). We then used the Illumina TruSeq RNA library preparation kit as per manufacturer’s instructions (Illumina Inc., CA, USA). The libraries were validated on a Bioanalyzer 2100 (Agilent Techonolgies, CA, USA), pooled and sequenced on two lanes of the Illumina HiSeq 2000 platform at the Biomolecular Resource Facility at the Australian National University, using a 150 bp paired-end run (all sequences were uploaded to the SRA database under the Bioproject ID: PRJNA388506).