small RNA-Seq of cerebrospinal fluid from patient with brain metastasis
CSF samples collected for the study were immediately centrifuged (500  g, 10 min, 4C) after the collection to separate higher-density particles and contaminants from the supernatant, which was then transferred to new tubes and frozen at 80C until further use. RNA isolation and purification: After thawing CSF on ice, 1 ml of sample was used for isolation and purification of total RNA enriched for small RNA species using the Urine microRNA Purification Kit (Norgen Biotek Corp., Canada) according to the manufacturer's instructions. Purified RNA was then frozen at 80C until further use. Library preparation, pooling, and sequencing: Small RNA libraries were constructed from total RNA using QIAseq miRNA Library Kit (Qiagen) according to the manufacturer's instructions. The RNA input was 5 l for CSF. Subsequently, libraries were normalized and pooled in equimolar ratio using online weight to molar quantity converter. Library pools (24 libraries in each pool) were then processed according to the NextSeq System Denature and Dilute Libraries Guide. Denatured and diluted PhiX Control v3 was added at 1% to all pools as an internal standard and single-read sequencing with 75 bp read length was performed using NextSeq 500 Sequencing System and NextSeq 500/550 High Output v2 kit (75 cycles) (all Illumina, USA). Approximately 16.7 M sequencing reads per library were expected.