Qiaseq mirna seq library preparation kit

MicroRNA sequencing was conducted as previously described.^{1 (link)} Briefly, cell-free total RNA was isolated from 200 µL of CSF using the Qiagen miRNAeasy serum/plasma extraction kit (Qiagen, 217184). Cursory Quality control analysis to confirm RNA was performed on each sample using UV-VIS spectroscopy on a Nanodrop ND-1000 (ThermoFisher). As per the manufacturer's instructions, samples were normalized to an input volume of 5 μL RNA eluate to prepare miRNAseq libraries using the Qiagen QiaSeq miRNA-seq library preparation kit (Qiagen, 331502). Next-generation sequencing was performed on a NextSeq 550 Sequencing System (Illumina) using single-end (SE) 1 × 75 base pairs sequencing to a depth of 25 million raw reads per sample. FASTQ files are uploaded to the online Qiagen Data Analysis Center for miRNA quantification. In the primary QIAseq quantification step, the Unique Molecular Identifier (UMI) counts are calculated, and primary miRNA mapping was performed using a human-specific miRBase mature database. In the primary QIAseq quantification step, adapter sequences from the library preparation process and any low-quality bases are removed. UMI counts for each miRNA were used for differential expression analysis.