This protocol details the RNAseq sample preparation starting with mRNA and ending with quality control of finished libraries. This protocol was used specifically for virus mRNA samples related to ORFeome lab group.
Method Article
RNAseq: mRNA to Illumina library
https://doi.org/10.1038/protex.2015.013
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This protocol details the RNAseq sample preparation starting with mRNA and ending with quality control of finished libraries. This protocol was used specifically for virus mRNA samples related to ORFeome lab group.
This protocol details the RNAseq sample preparation starting with mRNA. This is a modification of the TruSeq Stranded mRNA Sample Preparation since the published protocol usually starts with Total RNA instead of mRNA. Protocol also includes assays used for quality control of samples.
Agilent RNA 6000 Nano Kit (5067-1511)
Agilent High Sensitivity DNA Kit (5067-4626)
Illumina Tru Seq Stranded mRNA Kit Set A (RS-122-2101)
Quant-iT™ PicoGreen® dsDNA Assay Kit (P7589)
2100 Electrophoresis Bioanalyzer Instrument (G2939AA)
Fluorescence plate reader
b. Following procedure requires 10-400ng of mRNA for each sample.
i. Illumina recommends having mRNA concentrated into 5 µL or less to start; however, we have had success with using up to 42 µL, equal to 400ng of mRNA.
b. Proceed with incubation.
c. Since starting with mRNA, without beads, skip page 22 step #2. At step #3 only transfer 17 µL of mRNA/mix and continue protocol as Illumina has written.
b. Assess size of library using Agilent High Sensitivity DNA Kit (Supplementary 3) . Refer to the Quick Guide. The final product should be approximately 260bp.
Agilent RNA 6000 Nano-1.5hrs
TruSeq Stranded mRNA Prep-9hrs
Agilent HS DNA-1.5hrs
Picogreen-0.5hrs
Please refer troubleshooting to applicable commercial company technical support.
A valid Illumina Library to send for sequencing.
UNC High-Throughput Sequencing Facility for sequencing the finished libraries.
None
Supplementary 1 Agilent RNA 6000 Nano Quick Guide Agilent RNA 6000 Nano Kit Quick Start Guide-Rev.C. (2013) Available at: http://www.chem.agilent.com/library/usermanuals/Public/G2938-90037_RNA6000Nano_QSG.pdf.
Supplementary 3 Agilent High-Sensitivity DNA Quick Guide Agilent High Sensitivity DNA Kit Quick Start Guide-Rev.C. (2013) Available at: http://www.chem.agilent.com/library/usermanuals/Public/G2938-90322_HighSensitivityDNA_QSG.pdf.
Supplementary 2 Picogreen dsDNA Assay Quant-iT™ PicoGreen ® dsDNA Reagent and Kits-Rev.10-June-2008. (2008) Available at: http://tools.lifetechnologies.com/content/sfs/manuals/mp07581.pdf.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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