Total RNA was extracted using the NucleoSpin RNA Mini kit (Macherey-Nagel). Initial RNA integrity numbers (RIN) were measured with an Agilent 4200 Tapestation instrument. RIN values ranged from 8.4 to 10.0 with a mean of 9.9. Libraries were prepared using the TruSeq Stranded Total RNA Gold kit, and sequencing was carried out according to Illumina protocols at the Northwest Genomics Center of the University of Washington. Massively parallel sequencing-by-synthesis with fluorescently labeled, reversibly terminating nucleotides was carried out on a NovaSeq 6000 instrument to provide 100bp paired-end fragments. Resulting FASTQ files were aligned to GENCODE v29(21 (link)) using STAR (v2.6.1d)(22 (link)) and RSEM (v1.3.1)(23 (link)). A median mapping rate of 94% was observed, yielding 22.5 uniquely mapped reads per sample.
Protocol detail
High-Quality RNA Sequencing Pipeline
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Corresponding Organization : University of Virginia
Other organizations : University of Southern California, Fred Hutch Cancer Center, University of Virginia Health System
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Variable analysis
independent variables
- None explicitly mentioned
- RNA integrity numbers (RIN)
- Mapping rate
- Uniquely mapped reads per sample
- None explicitly mentioned
- Positive control: None mentioned
- Negative control: None mentioned
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