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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present a protocol optimized for the processing of coding (mRNA) and non-codingĀ (ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

Abstract

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omittedĀ size selection, to allow capture of more RNA species.

Introduction

Next generation sequencing (NGS) has emerged as a powerful tool for the investigation of the changes that occur at the genomic level of biological organisms. Sample preparation for NGS methods can be varied depending on the organism, tissue type, and more importantly the questions the researchers are keen to address. Many studies have turned to NGS as a means of studying the differences in gene expression between states such as healthy and sick individuals1,2,3,4. The sequencing take place on a whole genome basis and allows a researcher to c....

Protocol

Animal protocols were approved by the National Animal Disease Center (USDA-ARS-NADC) Animal Care and Use Committee.

1. Collection of Swine Blood Samples

  1. Collect blood samples into RNA tubes. Collect ~2.5 mL or more if larger collection tubes are available.

2. Processing of Swine Blood Samples

  1. Centrifuge the blood tubes at 5,020 x g for 10 min at room temperature (15-25 Ā°C). If processing frozen samples incubate tube at ro.......

Representative Results

The representative samples in our study are the globin and ribo-depleted whole blood samples. The representative outcome of the protocol consists of a globin depleted library sample with an RNA integrity number (RIN) above 7 (Figure 1a) and 260/280 nm concentration ratios at or above 2 (Figure 1b and 1c). Validation of the sample outcome was performed using spectrophotometer to give the final con.......

Discussion

The first critical step in the protocol that made it optimized included the added globin depletion steps, which made it possible to get quality reads from whole blood samples. One of the largest limitations on using whole blood in sequencing studies are the high numbers of reads in the sample that will map to globin molecules and reduce the reads that could map to other molecules of interest18. Therefore, in optimizing the protocol for our sample type, we needed to incorporate a globin depletion s.......

Acknowledgements

This work was mainly supported by the by USDA NIFA AFRI 2013-67015-21236, and in part by USDA NIFA AFRI 2015-67015-23216. This study was supported in part by an appointment to the Agricultural Research Service Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the US Department of Energy (DOE) and the US Department of Agriculture. ORISE is managed by Oak Ridge Associated Universities under DOE contract no. DE-AC05-06OR2310.

We would like to thank Dr. Kay Faaberg for the HP-PRRSV infectious clones, Dr. Susan Brockmeier for her help with animals inv....

Materials

NameCompanyCatalog NumberComments
PAXgene TubesPreAnalytix762165
Molecular Biology Grade WaterThermoFisher10977-015
mirVana miRNA Isolation KitThermoFisherAM1560
Rneasy MinElute Clean Up KitQIAGEN74204
100% EthanolDecon Labs, Inc.2716
0.2 mL thin-walled tubesThermoFisher98010540
1.5 mL RNase/DNase - free tubesAny supplier
Veriti 96-well ThermocyclerThermoFisher4375786R
Globin Reduction Oligo (Ī± 1)Any supplierSequence GAT CTC CGA GGC TCC AGC TTA ACG GT
Globin Reduction Oligo (Ī± 2)Any supplierSequence TCA ACG ATC AGG AGG TCA GGG TGC AA
Globin Reduction Oligo (Ī² 1)Any supplierSequence AGG GGA ACT TAG TGG TAC TTG TGG GT
Globin Reduction Oligo (Ī² 2)Any supplierSequence GGT TCA GAG GAA AAA GGG CTC CTC CT
10X Oligo Hybridization Buffer
-Tris-HCl, pH 7.6Fisher ScientificBP1757-100
-KClMillipore Sigma60142-100ML-F
10X RNase H Buffer
Ā -Tris-HCl, pH 7.6Fisher ScientificBP1757-100
Ā -DTTThermoFisherY00147
Ā -MgCl2PromegaA351B
Ā -Molecular Biology Grade WaterThermoFisher10977-015
RNase HThermoFisherAM2292
SUPERase-INThermoFisherAM2694Rnase inhibitor
EDTAMillipore SigmaE7889
MicrocentrifugeAny supplier
Ā 2100 Electrophoresis BioAnalyzer InstrumentAgilent TechnologiesG2938C
Agilent RNA 6000 Nano KitAgilent Technologies5067-1511
Agilent High Sensitivity DNAĀ  KitAgilent Technologies5067-4626
TruSeq Stranded Total RNA Library Prep Kit with Ribo-ZeroĀ IlluminaRS-122-2201mRNA kit; Human/Mouse/Rat Set AĀ  (48 samples, 12 indexes)
TruSeq Stranded Total RNA Sample Preparation GuideIlluminaAvailable on-line
RNAClean XP BeadsBeckmanCoulterA63987
AMPure XP BeadsBeckmanCoulterA63880
MicroAmp Optical 8-tube StripThermoFisherN80105800.2 ml thin-walled tubes
MicroAmp Optical 8-tube Strip CapThermoFisherN801-0535
RNase/DNase - free reagent reservoirsAny supplier
SuperScript II Reverse TranscriptaseThermoFisher18064-014
MicroAmp Optical 96 well platesThermoFisherN8010560These were used in place of .3mL plates as needed
MicroAmp Optical adhesive filmThermoFisher4311971
NEBNext Multiplex Small RNA Library Prep Set for IlluminaĀ® (Set 1)Ā New England BiolabsE73005small RNA kit
NEBNext Multiplex Small RNA Library Prep Set for IlluminaĀ® (Set 2)Ā New England BiolabsE75805small RNA kit
QIAQuick PCR Purification KitQIAGEN28104
96S Super Magnet PlateALPAQUAA001322

References

  1. Finotello, F., Di Camillo, B. Measuring differential gene expression with RNA-seq: challenges and strategies for data analysis. Briefings in Functional Genomics. 14 (2), 130-142 (2015).
  2. Coble, D. J., et al.

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