Depletion of Ribosomal RNA Sequences from Single‐Cell RNA‐Sequencing Library

Nan Fang1, Rumeysa Akinci‐Tolun1

1 QIAGEN GmbH, Hilden
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.27
DOI:  10.1002/cpmb.11
Online Posting Date:  July, 2016
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Abstract

Recent advances in single‐cell RNA sequencing technologies have revealed high heterogeneity of gene expression profiles in individual cells. However, most current single‐cell RNA‐seq methods use oligo‐dT priming in the reverse transcription steps and detect only polyA‐positive for more accuracy, since there are also polyA‐positive non‐coding RNAs transcripts, not other important RNA species, such as polyA‐negative noncoding RNA. Reverse transcription using random oligos enables detection of not only the noncoding RNA species without polyA tails, but also ribosomal RNA (rRNA). rRNA comprises more than 90% of the total RNA and should be depleted from the RNA‐seq library to ensure efficient usage of the sequencing capacity. Commonly used hybridization‐based rRNA depletion methods can preserve noncoding RNA in the standard RNA‐seq library. However, such rRNA depletion methods require high input amounts of total RNA and do not work at the single‐cell level or with limited input DNA. This unit describes a novel procedure for RNA‐seq library construction from single cells or a minimal amount of RNA. A thermostable duplex‐specific nuclease is used in this method to effectively remove ribosomal RNA sequences following whole‐transcriptome amplification and sequencing library construction. © 2016 by John Wiley & Sons, Inc.

Keywords: ribosomal RNA depletion; RNA‐seq library construction; single cell RNA sequencing; whole transcriptome amplification

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Repli‐g Whole‐Transcriptome Amplification from Single Cells
  • Alternate Protocol 1: Repli‐g Whole Transcriptome Amplification from Purified RNA
  • Basic Protocol 2: Construct RNA‐Sequencing Library from Repli‐g –Amplified cDNA
  • Basic Protocol 3: DSN Digestion to Remove Ribosomal RNA Sequences from RNA‐Seq Library
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Repli‐g Whole‐Transcriptome Amplification from Single Cells

  Materials
  • Ice
  • Cell material (1‐1000 human or animal cells), suspended in 7 μl PBS
  • REPLI‐g WTA Single Cell Kit (QIAGEN, cat. no. 150063 or 150065), which contains the following reagents that have gone through special de‐contamination treatment to remove nucleic acid contamination so that the contaminating nucleic acid will not be amplified along with the cDNA and interfere with result interpretation:
    • Lysis buffer
    • NA Denaturation buffer
    • gDNA Wipeout buffer, WTA
    • RT/Polymerase buffer
    • Random primer
    • Oligo dT primer
    • Quantiscript RT Enzyme mix
    • Ligase mix
    • Ligase buffer
    • REPLI‐g Reaction buffer
    • REPLI‐g SensiPhi DNA polymerase
  • Nuclease‐free water
  • Pipets and pipet tips
  • Vortex mixer
  • 0.2‐ml and 1.5‐ml microcentrifuge tubes or PCR‐strips
  • Thermal cycler
  • Microcentrifuge that accommodates 0.2‐ml tubes or PCR‐strips (e.g., 16‐place microcentrifuge for 0.2‐ml tubes from USA Scientific)

Alternate Protocol 1: Repli‐g Whole Transcriptome Amplification from Purified RNA

  Materials
  • Ice
  • Purified RNA, 50 pg to 100 ng, suspended in 7 μl PBS
  • REPLI‐g WTA Single Cell Kit (QIAGEN, cat. no. 150063 or 150065), which contains the following reagents that have gone through special treatment to remove nucleic acid contamination so that the contaminating nucleic acid will not be amplified along with the cDNA and interfere with result interpretation:
    • Lysis buffer
    • NA Denaturation buffer
    • gDNA Wipeout buffer, WTA
    • RT/Polymerase buffer
    • Random primer
    • Oligo dT primer
    • Quantiscript RT Enzyme mix
    • Ligase mix
    • Ligase buffer
    • REPLI‐g Reaction buffer
    • REPLI‐g SensiPhi DNA polymerase
  • Nuclease‐free water
  • Vortex mixer
  • Pipets and pipet tips
  • 1.5‐ml and 0.2‐ml microcentrifuge tubes or PCR‐strips
  • Microcentrifuge that accommodates 0.2‐ml tubes or PCR‐strips (e.g., 16‐place microcentrifuge for 0.2 ml tubes from USA Scientific)
  • Thermal cycler

Basic Protocol 2: Construct RNA‐Sequencing Library from Repli‐g –Amplified cDNA

  Materials
  • REPLI‐g ‐amplified double‐stranded cDNA (see protocol 1 or Alternate Protocol 1)
  • RNase‐free water
  • 80% ethanol, Buffer TE
  • GeneRead DNA Library I Core kit (QIAGEN, cat. no. 180432 or 180434) containing:
    • End Repair Buffer, 10×
    • End Repair Enzyme Mix
    • A‐Addition Buffer, 10×
    • Klenow Fragment
    • Ligation Buffer, 2×
    • T4 DNA Ligase
    • RNase‐free H 2O
  • GeneRead Adapters (QIAGEN, cat. no. 180985 or 180986), set of 12 ready‐to‐use sequencing adapters with unique indices
  • Ice
  • GeneRead Size Selection Kit (Qiagen cat. no. 180514) containing:
    • Buffer SB1
    • Buffer EB
    • MinElute Spin Columns
    • 2‐ml collection tubes
  • GeneRead DNA I Amp Kit (Qiagen cat. no. 180455) containing:
    • HiFi PCR Master Mix, 2×
    • Library Amplification Primer mix
  • Instrument for DNA shearing (e.g., Covaris Focused‐ultrasonicator)
  • Microcentrifuge
  • 1.5‐ml microcentrifuge tubes
  • Instrument to quantify purified, sheared cDNA [e.g., QIAxpert (QIAGEN) or Qubit Flurometer (Thermo Fisher Scientific]
  • DNA LoBind tubes (from Axygene or Eppendorf)
  • PCR tubes or plates
  • Pipet tips and pipets
  • Vortex mixer
  • Thermal cycler
  • 2100 Bioanalyzer (Agilent) for RNA‐seq Library QC

Basic Protocol 3: DSN Digestion to Remove Ribosomal RNA Sequences from RNA‐Seq Library

  Materials
  • 1 M HEPES buffer (Sigma Aldrich, cat. no. 83264)
  • 5 M NaCl solution
  • RNase‐free water
  • Duplex‐specific nuclease (lyophilized) (including DSN enzyme, lyophilized, DSN storage buffer, 50 mM Tris·Cl, 10× DSN master buffer, and 2× DSN stop solution) (Evrogen, cat. no. EA001)
  • 100% glycerol
  • Ice
  • Sequencing library, 100 ng (see protocol 3)
  • MinElute PCR Purification Kit (QIAGEN, cat. no. 28004 and 28006) containing:
    • 2‐ml collection tubes
    • MinElute spin columns
    • Buffer PB
    • Buffer PE
    • Buffer EB
  • GeneRead DNA I Amp Kit (QIAGEN, cat. no. 180455) containing:
    • HiFi PCR Master Mix, 2×
    • Primer mix (10 μM each)
  • Microcentrifuge
  • PCR tubes or plates
  • Pipet tips and pipets
  • Two thermal cyclers with lids that can be opened during incubation, placed next to each other (e.g., Eppendorf Mastercycler)
  • 1.5‐ml microcentrifuge tubes
  • 2100 Bioanalyzer (Agilent)
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Figures

Videos

Literature Cited

Literature Cited
  Ben‐Ezra, J., Johnson, D.A., Rossi, J., Cook, N., and Wu, A. 1991. Effect of fixation on the amplification of nucleic acids from paraffin‐embedded material by the polymerase chain reaction. J. Histochem. Cytochem. 39:351‐354. doi: 10.1177/39.3.1704393.
  de Bourc, C.F.A., De Vlaminck, I., Kanbar, J.N., Wang, J., Gawad, C., and Quake, S.R. 2014. A quantitative comparison of single‐cell whole genome amplification methods. PLoS One 9:e10558. doi: 10.1371/journal.pone.0105585.eCollection 2014.
  Eberwine, J., Sul, J.Y., Bartfai, T., and Kim, J. 2014. The promise of single cell sequencing. Nat. Methods 11:25‐27. doi: 10.1038/nmeth.2769.
  Esteller, M. 2013. Non‐coding RNAs in human disease. Nat. Rev. Genet. 12:861‐874. doi: 10.1038/nrg3074.
  Fatica, A. and Bozzoni, I. 2013. Long non‐coding RNAs: New players in cell differentiation and development. Nat. Rev. Genet. 15:7‐21. doi: 10.1038/nrg3606.
  Han, L., Zi, X.Y., Garmire, L.X., Wu, Y., Pan, X.H., Weissman, S.M., and Fan, R. 2014. Co‐detection and sequencing of genes and transcripts from the same single cells facilitated by a microfluidics platform. Sci. Rep. 4:6485. doi: 10.1038/srep06485.
  Korfhage, C., Fricke, E., and Meier, A. 2015. Whole‐transcriptome amplification of single cells for next‐generation sequencing. Curr. Protoc. Mol. Biol. 111:7.20.1‐7.20.19. doi: 10.1002/0471142727.mb0720s111.
  Kornienk, A.E., Guenz, P.l., Barlow, D.P., and Paule, F.M. 2013. Gene regulation by the act of long non‐coding RNA transcription. BMC Biol. 11:59. doi: 10.1186/1741‐7007‐11‐59.
  Macaulay, I.C. and Voet, T. 2014. Single cell genomics: Advances and future perspectives. PLoS Genet. 10:e1004126. doi: 10.1371/journal.pgen.1004126.
  Nagalakshmi, U., Waern, K., and Snyder, M. 2010. RNA‐seq: A method for comprehensive transcriptome analysis. Curr. Protoc. Mol. Biol. 89:4.11.1‐4.11.13.
  Nelson, J.R. 2014. Random‐primed, Phi29 DNA polymerase‐based whole genome amplification. Curr. Protoc. Mol. Biol. 105:15.13.1‐15.13.16.
  Sanberg, R. 2014. Entering the era of single‐cell transcriptomics in biology and medicine. Nat. Methods 11:22‐24. doi: 10.1038/nmeth.2764.
  Shagin, D.A., Rebrikov, D.V., Kozhemyako, V.B., Altshuler, I.M., Shcheglov, A.S., Zhulidov, P.A., Bogdanova, E.A., Staroverov, D.B., Rasskazov, V.A., and Lukyanov, S. 2002. A novel method for SNP detection using a new duplex‐specific nuclease from crab hepatopancreas. Genome Res. 12:1935‐1942. doi: 10.1101/gr.547002.
  Thompson, J.F. and Steinmann, K.E. 2010. Single molecule sequencing with a HeliScope genetic analysis system. Curr. Protoc. Mol. Biol. 92:7.10.1‐7.10.14.
  Van Ooyen, S., Loeffert, D., and Korfhage, C. 2011. Overcoming constraints of genomic DNA isolated from paraffin‐embedded tissue. http://www.qiagen.com/Products/Catalog/Sample‐Technologies/DNA‐Sample‐Technologies/Genomic‐DNA/REPLI‐g‐FFPE‐Kit#resources.
  Veitia, R.A. 2008. One thousand and one ways of making functionally similar transcriptional enhancers. BioEssays 30:1052‐1057. doi: 10.1002/bies.20849.
  Wu, A.R., Neff, N.F., Kalisky, T., Dalerba, P., Treutlein, B., Rothenberg, M.E., Mburu, F.M., Mantalas, G.L., Sim, S., Clarke, M.F., and Quake, S.R. 2014. Quantitative assessment of single‐cell RNA‐sequencing methods. Nat. Methods 11:41‐46. doi: 10.1038/nmeth.2694.
  Yi, H., Cho, Y.J., Won, S., Lee, J.E., Jin Yu, H., Kim, S., Schroth, G.P., Luo, S., and Chun, J. 2011. Duplex‐specific nuclease efficiently removes rRNA for prokaryotic RNA‐seq. Nucleic Acids Res. 39:e140. doi: 10.1093/nar/gkr617.
  Zhao, W., He, X., Hoadley, K.A., Parker, J.S., Hayes, D.N., and Perou, C.M. 2014. Comparison of RNA‐Seq by poly (A) capture, ribosomal RNA depletion, and DNA microarray for expression profiling. BMC Genomics 15:419. doi: 10.1186/1471‐2164‐15‐419.
  Zhu, Y.Y., Machleder, E.M., Chenchik, A., Li, R., and Siebert, P.D. 2001. Reverse transcriptase template switching: A SMART approach for full‐length cDNA library construction. Biotechniques 30:892‐897.
  Zhulidov, P.A., Bogdanova, E.A., Shcheglov, A.S., Vagner, L.L., Khaspekov, G.L., Kozhemyako, V.B., Matz, M.V., Meleshkevitch, E., Moroz, L.L., Lukyanov, S.A., and Shagin, D.A., 2004. Simple cDNA normalization using kamchatka crab duplex‐specific nuclease. Nucleic Acids Res. 32:e37. doi: 10.1093/nar/gnh031.
Internet Resources
  https://www.qiagen.com/de/resources/resourcedetail?id=f2430d44‐7f49‐4544‐b2de‐5a51b904ea39&lang=en.
  Handbook for REPLI‐g Single Cell WTA Kit (Qiagen).
  https://www.qiagen.com/de/resources/resourcedetail?id=014953d1‐be52‐4385‐baaa‐cd8446506404&lang=en.
  Handbook for GeneRead DNA Library I Core Kit (Qiagen).
  http://www.evrogen.com/protein‐descriptions/UM‐DSN.pdf.
  Handbook for duplex‐specific nuclease (Evrogen).
  https://support.illumina.com/content/dam/illumina‐support/documents/myillumina/7836bd3e‐3358‐4834‐b2f7‐80f80acb4e3f/dsn_normalization_sampleprep_application_note_15014673_c.pdf.
  RNA Analysis Application Note from Illumina: ‘DSN Normalization: Application of Duplex‐Specific Thermostable Nuclease (DSN) to normalize RNA samples for Illumina Sequencing’.
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