small RNA-Seq of blood plasma obtained from patient with brain metastasis
Peripheral blood samples collected for the study were centrifuged (2000  g, 10 min, 4C) immediately after collection to obtain blood plasma, which was separated, transferred to clean tubes, and then frozen at 80C until further use. RNA isolation and purification: After thawing plasma samples on ice, 250 l of the sample were transferred to a clean tube and centrifuged (1000  g, 5 min, 4C). Subsequently, 200 l of supernatant were used for isolation of total RNA enriched for small RNA species using the miRNeasy Serum/Plasma Kit (Qiagen, Germany) 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 samples using QIAseq miRNA Library Kit (Qiagen) according to the manufacturer's instructions. The RNA input was 5 l for plasma. 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.