human:cerebrospinal fluid
For viral metagenomic analysis, cerebrospinal fluid (CSF) samples were collected from 10 patients with encephalitis of unknown etiology in 2023. The 10 CSF samples were randomly pooled into a single specimen pool. This pooled sample was resuspended in 1 mL DPBS and subjected to three rapid freeze-thaw cycles on dry ice. After centrifugation (10 minutes, 15,000 g), the supernatant was collected and filtered through a 0.45 uL membrane (Millipore) to remove eukaryotic and bacterial cell-sized particles. From the filtered supernatant, 200 uL was treated with a nuclease cocktail to reduce free (non-viron-protected) nucleic acids. Total nucleic acids were extracted from the treated sample using the QIAamp MinElute Virus Spin Kit per the manufacturer's protocol. A single library was prepared with the Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced on the MiSeq Illumina platform, generating 250 bases paired ends with dual barcoding for the pool.