RNA metagenomic sequencing of Haemaphysalis longicornis metagenome: viral analysis from tick homogenate in Shandong, China
Total RNA was extracted from homogenized Haemaphysalis longicornis ticks collected from agricultural land in Weifang, Shandong, China, using the TRIzol Reagent (Invitrogen) followed by DNase I treatment to remove residual DNA. RNA integrity was verified by Bioanalyzer 2100 (Agilent Technologies, RIN > 7.0).  Ribosomal RNA was not depleted to retain potential viral RNA. The RNA was reverse-transcribed into double-stranded cDNA using random hexamer primers and the NEBNext Ultra II RNA Library Prep Kit (NEB). Fragmentation and adapter ligation were performed according to the manufacturers protocol.  Libraries were quantified by Qubit Fluorometer (Thermo Fisher) and pooled for sequencing on the Illumina NovaSeq 6000 platform with 150-bp paired-end reads. No PCR amplification or viral-specific probes were used to ensure unbiased detection of viral sequences.