Ovation rnaseq ffpe system

RNASeq was used to analyse the miRNA and mRNA expression in each CSF pool. Small RNASeq was performed using 3.67 ng of RNA in the Illumina TruSeq small RNA sample library preparation kit (RS-200-0012), as previously reported [7 (link)], with reagents used in a half-reaction. Samples were assigned one of 48 possible indices, and went through 16 PCR cycles. Indexed samples were run on a gel and purified away from the adaptor band. The samples were then pooled and placed on a single-read Illumina V3 flowcell (GD-401-3001). Long RNASeq was performed using 2 ng of RNA in a NuGen Ovation RNASeq FFPE system (7150) for cDNA synthesis and RNA amplification. The samples were quantified using the Qubit dsDNA HS Assay Kit (Q3285; ThermoFisher), then moved forward to a KAPA Hyper Library Preparation Kit (KK8502; KAPA Biosystems). Each sample was assigned one of eight possible indices after one cycle of PCR and final libraries were quantified using a KAPA SYBR FAST Universal qPCR Kit (KK4824; KAPA Biosystems). Pooled and paired libraries were placed on a Paired End Illumina V3 flowcell (FPE-401-3001; Illumina). A threshold value of ≥5 counts per RNA was used as the cut-off value for inclusion in the small (miRNA) and long (mRNA) RNASeq data analysis.