small RNA-Seq of brain metastasis tissue
Fresh tissue samples were collected for the study and immediately stored in RNAlater Stabilization Solution (Thermo Fisher Scientific, USA) at 4C for 24 h after the collection and then frozen at 80C until further use. All tissue samples were histologically diagnosed according to the WHO 2021 classification scheme independently by two histopathologists. RNA isolation and purification: Total RNA enriched for small RNA species from fresh-frozen tissue samples was isolated using mirVana miRNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. 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 100 ng for tissue. 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.