Characterization of Plant Small RNAs by Next Generation Sequencing

Sandra M. Mathioni1, Atul Kakrana2, Blake C. Meyers3

1 Donald Danforth Plant Science Center, Saint Louis, Missouri, 2 Center for Bioinformatics and Computational Biology, University of Delaware, Newark, Delaware, 3 Division of Plant Sciences, University of Missouri, Columbia, Missouri
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20043
Online Posting Date:  March, 2017
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Abstract

Plant small RNAs are ∼20 to 24 nucleotide noncoding RNAs that typically have repressive regulatory roles in gene expression, functioning at the transcriptional or post‐transcriptional level. This influence on regulation of developmental and physiological processes has direct effects on phenotype. High‐throughput sequencing technologies have enabled the sequencing of millions of small RNAs. Along with decreased sequencing costs, recent improvements in small RNA library construction have facilitated the ability to use minimal amounts of input RNA for analysis. This unit describes steps to isolate total RNA from limited amounts of plant tissue to construct small RNA libraries and perform small RNA data processing. © 2017 by John Wiley & Sons, Inc.

Keywords: sRNA; plant RNA isolation; plant sRNA processing; maize; anthers

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Total RNA Isolation from Limited Samples
  • Alternate Protocol 1: Total RNA Isolation from Leaves
  • Basic Protocol 2: Size Selection of Small RNAs
  • Basic Protocol 3: Small RNA Library Construction
  • Basic Protocol 4: Small RNA Data Processing
  • Support Protocol 1: Setup of the Environment for Pre‐Processing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Total RNA Isolation from Limited Samples

  Materials
  • RNaseZap RNase decontamination solution (Thermo Fisher, Ambion, cat. no. AM9798)
  • Anther or other tissue from plant of interest
  • Liquid nitrogen
  • Plant RNA Reagent (Thermo Fisher, cat. no. 12322012), 4°C
  • 5 M NaCl (Thermo Fisher, Ambion cat. no. AM9760G)
  • Chloroform, molecular grade (Fisher Scientific, cat. no. BP1145‐1)
  • Glycoblue co‐precipitant (Ambion, cat. no. AM9516, 15 mg/ml)
  • Isopropanol, molecular grade (Fisher Scientific, cat. no. BP2618500), 4°C
  • Absolute ethanol, molecular grade (Fisher Scientific, cat. no. BP2818‐4), 4°C
  • Nuclease‐free DEPC‐treated water, molecular grade (Thermo Fisher, cat. no. AM9906)
  • Qubit RNA BR Assay Kit (Thermo Fisher, cat. no. Q10210)
  • Mortar and pestle (VWR, cat. nos. 89038‐144 and 89038‐160)
  • Filtered micropipet tips, low retention, nuclease free, molecular grade (USA Scientific)
  • 2.0‐ml nuclease‐free microcentrifuge tubes (USA Scientific, cat. no. 1620‐2700)
  • Fluorospectrometer (e.g., DeNovix DS‐11 FX+ or Qubit 3.0)
  • Additional reagents and equipment for denaturing formaldehyde agarose gel
CAUTION: The RNA extraction reagent should be used in a properly ventilated chemical fume hood.

Alternate Protocol 1: Total RNA Isolation from Leaves

  Additional Materials (also see protocol 1)
  • Sea sand (EMD Millipore, extra pure, acid‐purified, autoclaved; Fisher Scientific, cat. no. M1077111000; optional)
  • Acidified phenol/chloroform (pH 4.5 with isoamyl alcohol, 125:24:1, molecular grade; Thermo Fisher, cat. no. AM9722)
  • Standard disposable spatula (Fisher Scientific, cat. no. 14‐955‐550)
CAUTION: The RNA extraction reagent should be used in a properly ventilated chemical fume hood.

Basic Protocol 2: Size Selection of Small RNAs

  Materials
  • Urea (Fisher Scientific, cat. no. BP169‐212)
  • 40% (w/v) acrylamide solution (19:1 acrylamide/bisacrylamide, Thermo Fisher, cat. no. AM9024)
  • 10× TBE (VWR, cat. no. 97062‐118)
  • Nuclease‐free DEPC‐treated water, molecular grade (Thermo Fisher, cat. no. AM9906)
  • RNaseZap RNase decontamination solution (Thermo Fisher, Ambion, cat. no. AM9798)
  • 10% (w/v) ammonium persulfate (APS; see recipe)
  • TEMED (N,N,N′,N′−tetramethylethylenediamine, Sigma, cat. no. T9281)
  • 6× Blue/Orange Loading Dye (Promega, cat. no. G1881)
  • Formamide (≥99.5%, Fisher Scientific, cat. no. BP228‐100)
  • 10‐ and 25‐bp DNA ladders (10‐ and 25‐bp DNA Step Ladders, Promega, cat. nos. G4471 and G4511)
  • Total RNA sample (see protocol 1 or protocol 2Alternate Protocol)
  • GelRed nucleic acid stain, 10,000× (Phoenix Research Products, cat. no. RGB‐4103)
  • 0.3 M NaCl (prepared from 5 M stock [Thermo Fisher, Ambion cat. no. AM9760G] using nuclease‐free water)
  • Glycoblue co‐precipitant (Ambion, cat. no. AM9516, 15 mg/ml)
  • 3 M sodium acetate (NaOAc, Ambion, cat. no. AM9740)
  • Absolute ethanol, molecular grade (Fisher Scientific, cat. no. BP2818‐4), −20°C
  • 75% (v/v) ethanol (prepared using nuclease‐free water), −20°C
  • Qubit RNA BR Assay Kit (Thermo Fisher, cat. no. Q10210)
  • 50‐ml conical tube
  • Rocking, horizontal, and rotating shakers
  • Mini‐PROTEAN Tetra Cell, 1.5 mm, with 10‐well comb (Bio‐Rad, cat. no. 1658001EDU)
  • Millipore Nylon‐Net Steriflip Vacuum Filter Unit (Thermo Fisher, cat. no. SCNY00020)
  • Filtered micropipet tips, low retention, nuclease free, molecular grade (USA Scientific)
  • 10‐ml pipet with electronic dispenser
  • Nuclease‐free, low‐retention PCR tubes: 0.2‐ml (Thermo Fisher, Axygen cat. no. 14‐222‐251) or 1.7‐ml (VWR, GeneMate cat. no. 490004‐436)
  • Thermocycler or dry bath set at 65°C
  • Nuclease‐free microcentrifuge tubes: 0.5‐ml (Thermo Fisher, Axygen cat. no. 14‐222‐292) and 2.0‐ml (USA Scientific, cat. no. 1620‐2700)
  • Sterile 18‐G needle (BD, cat no. 305196)
  • Single‐edge razor blade (Fisher Scientific, cat. no. 12‐640)
  • Mini‐Blotting staining container (RPI, cat. no. 248717)
  • Gel imaging system
  • UV transilluminator
  • 0.45‐µm COSTAR Spin‐X filters (Thermo Fisher, cat. no. 07‐200‐387)
  • Fluorospectrometer (e.g., DeNovix DS‐11 FX+ or Qubit 3.0)

Basic Protocol 3: Small RNA Library Construction

  Materials
  • TruSeq Small RNA Library Prep Kit, Set A (Illumina, cat. no. RS‐200‐0012), including:
    • RNA 3′ and 5′ Adapters (RA3 and RA5)
    • Ligation Buffer (HML)
    • RNase Inhibitor
    • Stop Solution (STP)
    • 10 mM ATP
    • T4 RNA Ligase
    • 25 mM dNTP Mix
    • RNA RT Primer (RTP)
    • 5× First‐Strand Buffer
    • 100 mM DTT
    • PCR Mix
    • RNA PCR Primer (RP1)
    • RNA PCR Primer Indexes 1‐12 (RPIX)
  • Size‐selected small RNA sample (see protocol 3)
  • Nuclease‐free DEPC‐treated water, molecular grade (Thermo Fisher, cat. no. AM9906)
  • SuperScript II Reverse Transcriptase (Thermo Fisher, cat. no. 18064014)
  • T4 RNA Ligase 2, Deletion Mutant (Epicentre, cat. no. LR2D11310K)
  • RNaseZap RNase decontamination solution (Thermo Fisher, Ambion, cat. no. AM9798)
  • 40% (w/v) acrylamide solution (19:1 acrylamide/bisacrylamide, Thermo Fisher, cat. no. AM9024)
  • 10× TBE (VWR, cat. no. 97062‐118)
  • 10% (w/v) ammonium persulfate (APS; see recipe)
  • TEMED (N,N,N′,N′‐tetramethylethylenediamine, Sigma, cat. no. T9281)
  • 6× Blue/Orange Loading Dye (Promega, cat. no. G1881)
  • High‐resolution RNA ladder
  • Custom RNA ladder
  • GelRed nucleic acid stain, 10,000× (Phoenix Research Products, cat. no. RGB‐4103)
  • 0.3 M NaCl (prepared from 5 M stock [Thermo Fisher, Ambion cat. no. AM9760G] using nuclease‐free water)
  • Glycoblue co‐precipitant (Ambion, cat. no. AM9516, 15 mg/ml)
  • 3 M sodium acetate (NaOAc)
  • Absolute ethanol, molecular grade (Fisher Scientific, cat. no. BP2818‐4), −20°C
  • 70% (v/v) ethanol (prepared using nuclease‐free water), −20°C
  • Qubit dsDNA HS Assay Kit (Thermo Fisher, cat. no. Q32854)
  • Thermocycler
  • Filtered micropipet tips, nuclease‐free, molecular grade (USA Scientific)
  • 0.2‐ml nuclease‐free PCR tubes (Thermo Fisher, Axygen cat. no. 14‐222‐251)
  • Nuclease‐free microcentrifuge tubes:
  • 0.5‐ml (Thermo Fisher, Axygen cat. no. 14‐222‐292)
  • 1.7‐ml (VWR, GeneMate cat. no. 490004‐436)
  • 2.0‐ml (USA Scientific, cat. no. 1620‐2700)
  • Mini‐PROTEAN Tetra Cell, 1.5 mm, with 10‐well comb (Bio‐Rad, cat. no. 1658001EDU)
  • 50‐ml conical tube
  • 10‐ml pipet with electronic dispenser
  • Sterile 18‐G needle (BD, cat no. 305196)
  • Single‐edge razor blade (Fisher Scientific, cat. no. 12‐640)
  • Mini‐Blotting staining container (RPI, cat. no. 248717)
  • Horizontal shaker
  • Gel imaging system
  • UV transilluminator
  • 0.45‐µm COSTAR Spin‐X filters (Thermo Fisher, cat. no. 07‐200‐387)
  • Fluorospectrometer (e.g., DeNovix DS‐11 FX+ or Qubit 3.0)
  • Fragment analyzer (Advanced Analytical) or bioanalyzer (Agilent Technologies)
NOTE: Reagents should be thawed at the beginning of each specific procedure, as indicated in the steps below. All enzymes should be taken from the −20°C freezer immediately before use and returned to the freezer immediately after use.

Basic Protocol 4: Small RNA Data Processing

  Materials
  • Computing node or desktop computer with at least four computing cores and 16 GB RAM, running a UNIX‐based operating system, with internet access
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Figures

Videos

Literature Cited

  Axtell, M.J. 2013. ShortStack: Comprehensive annotation and quantification of small RNA genes. RNA 19:740‐751. doi: 10.1261/rna.035279.112.
  Baran‐Gale, J., Lisa Kurtz, C., Erdos, M.R., Sison, C., Young, A., Fannin, E.E., Chines, P.S., and Sethupathy, P. 2015. Addressing bias in small RNA library preparation for sequencing: A new protocol recovers microRNAs that evade capture by current methods. Front. Genet. 6:1‐9. doi: 10.3389/fgene.2015.00352.
  Baulcombe, D.C. 2015. VIGS, HIGS and FIGS: Small RNA silencing in the interactions of viruses or filamentous organisms with their plant hosts. Curr. Opin. Plant Biol. 26:141‐146. doi: 10.1016/j.pbi.2015.06.007.
  Borges, F. and Martienssen, R.A. 2015. The expanding world of small RNAs in plants. Nat. Rev. Mol. Cell Biol. 16:1‐15. doi: 10.1038/nrm4085.
  Brousse, C., Liu, Q., Beauclair, L., Deremetz, A., Axtell, M.J., and Bouché, N. 2014. A non‐canonical plant microRNA target site. Nucleic Acids Res. 42:5270‐5279. doi: 10.1093/nar/gku157.
  Chen, X. 2009. Small RNAs and their roles in plant development. Ann. Rev. Cell Dev. Biol. 35:21‐44. doi: 10.1146/annurev.cellbio.042308,113417.
  Jones‐Rhoades, M.W., Bartel, D.P., and Bartel, B. 2006. MicroRNAs and their regulatory roles in plants. Ann. Rev. Plant Biol. 57:19‐53. doi: annurev.arplant57.032905.105218.
  Kakrana, A., Hammond, R., Patel, P., Nakano, M., and Meyers, B.C. 2014. SPARTA: A parallelized pipeline for integrated analysis of plant miRNA and cleaved mRNA data sets, including new miRNA target‐identification software. Nucleic Acids Res. 42:1‐13. doi: 10.1093/nar/gku693.
  Kozomara, A. and Griffiths‐Jones, S. 2014. miRBase: Annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res. 42:D68‐73. doi: 10.1093/nar/gkt1181.
  Lu, C., Tej, S.S., Luo, S., Haudenschild, C.D., Meyers, B.C., and Green, P.J. 2005. Elucidation of the small RNA component of the transcriptome. Science 309:1567‐1569. doi: 10.1126/science.1114112.
  Nobuta, K., Lu, C., Shrivastava, R., Pillay, M., De Paoli, E., Accerbi, M., Arteaga‐Vazquez, M., Sidorenko, L., Jeong, D.H., Yen, Y., Green, P.J., Chandler, V.L., and Meyers, B.C. 2008. Distinct size distribution of endogenous siRNAs in maize: Evidence from deep sequencing in the mop1‐1 mutant. Proc. Natl. Acad. Sci. U.S.A. 105:14958‐14963. doi: 10.1073/pnas.0808066105.
Internet Resources
  www.mirbase.org
  miRBase: The microRNA Database. Provides a searchable database of published miRNA sequences.
  mpss.danforthcenter.org
  Next‐Gen Sequence Databases. Has small RNA data publicly available for several plant species.
  https://phytozome.jgi.doe.gov/pz/portal.html
  Phytozome: The Plant Genomics Resource. A Plant Comparative Genomics Portal with 65 sequenced and annotated plant genomes.
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