Viruses Microbiology Genetics and Genomics

scientificprotocols authored almost 3 years ago

Authors: Marcia Sanders & Anne Beall

Abstract

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.

Introduction

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.

Reagents

  1. Agilent RNA 6000 Nano Kit (5067-1511)
  2. Agilent High Sensitivity DNA Kit (5067-4626)
  3. Illumina Tru Seq Stranded mRNA Kit Set A (RS-122-2101)
  4. Quant-iT™ PicoGreen® dsDNA Assay Kit (P7589)

Equipment

  1. 2100 Electrophoresis Bioanalyzer Instrument (G2939AA)
  2. Fluorescence plate reader

Procedure

  1. Assess mRNA using Agilent RNA 6000 Nano kit, assay mRNA Nano (Supplementary 1).
    • a. Best if each mRNA sample run in duplicate so an average mRNA concentration can be used.
    • b. Following procedure requires 10-400ng of mRNA for each sample.
  2. Proceed with Illumina TruSeq Stranded mRNA Sample Preparation Guide, Low Sample (LS) Protocol (15031047 Revision E).
    • a. Since starting with mRNA, start at page 20 step #12 by adding mix to desired amount of mRNA.
      • 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.
  3. Validate library after complete.
    • a. Assess quantity of dsDNA library using Quant-iT™ PicoGreen® dsDNA Assay Kit (Supplementary 2). Minimum quantity will depend on laboratory/core the library will be submitted to.
    • 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.

Timing

  • Agilent RNA 6000 Nano-1.5hrs
  • TruSeq Stranded mRNA Prep-9hrs
  • Agilent HS DNA-1.5hrs
  • Picogreen-0.5hrs

Troubleshooting

Please refer troubleshooting to applicable commercial company technical support.

Anticipated Results

A valid Illumina Library to send for sequencing.

References

  1. 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.
  2. 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.
  3. Low Sample (LS) Protocol. TruSeq Stranded mRNA Sample Preparation Guide. [Revision E] 20-43
  4. Quant-iT™ PicoGreen ® dsDNA Reagent and Kits-Rev.10-June-2008. (2008) Available at: http://tools.lifetechnologies.com/content/sfs/manuals/mp07581.pdf.

Acknowledgements

UNC High-Throughput Sequencing Facility for sequencing the finished libraries.

Figures

Supplementary 1: Agilent RNA 6000 Nano Quick Guide

Download Supplementary 1

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 2: Picogreen dsDNA Assay

Download Supplementary 2

Quant-iT™ PicoGreen ® dsDNA Reagent and Kits-Rev.10-June-2008. (2008) Available at: http://tools.lifetechnologies.com/content/sfs/manuals/mp07581.pdf.

Supplementary 3: Agilent High-Sensitivity DNA Quick Guide

Download Supplementary 3

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.

Author information

Marcia Sanders & Anne Beall, ORFeome

Correspondence to: Marcia Sanders ([email protected]), Anne Beall ([email protected]

Source: Protocol Exchange (2015) doi:10.1038/protex.2015.013. Originally published online 13 February 2015.

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