Whole‐Transcriptome Amplification of Single Cells for Next‐Generation Sequencing

Christian Korfhage1, Evelyn Fricke1, Andreas Meier1

1 QIAGEN GmbH, Hilden
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.20
DOI:  10.1002/0471142727.mb0720s111
Online Posting Date:  July, 2015
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Abstract

Single‐cell transcriptome analysis gives insight into the heterogeneity of neighboring cells in tissues or cell cultures. It has been shown that the variability of cells is important for biological function. However, cell variability can be an indication of disease (e.g., cancer). In order to elucidate cell variability in health and disease, single‐cell transcriptomes can be analyzed by new next‐generation sequencing (NGS) platforms. NGS analysis currently requires a few hundred nanograms of RNA. Consequently, accurate whole‐transcriptome amplification is required to analyze the transcriptome of a single cell by NGS. This unit describes the entire workflow from cells to NGS, including cell quality testing, cell lysis, gDNA removal, whole‐transcriptome amplification, and NGS library preparation. Recommendations for WTA quality control are given and optional protocols for using purified RNA for WTA and WTA purification are described. © 2015 by John Wiley & Sons, Inc.

Keywords: transcriptome analysis; single cell; next‐generation sequencing (NGS); cancer; whole transcriptome amplification (WTA); gene regulation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Determination of the Fraction of Viable Cells with a Cell Sample
  • Basic Protocol 2: Preparation of Cell Samples by Dilution
  • Basic Protocol 3: LYSE Cell Samples
  • Alternate Protocol 1: Use of Purified RNA Samples Instead of Cells for Whole‐Transcriptome Amplification
  • Basic Protocol 4: Whole Transcriptome Amplification (WTA)
  • Basic Protocol 5: Use of WTA DNA for Next‐Generation Sequencing
  • Support Protocol 1: Quality Control of WTA DNA using Real‐Time PCR
  • Support Protocol 2: Optional Cleanup of WTA DNA using Agencourt Ampure XP Magnetic Beads
  • Support Protocol 3: Optional Cleanup of WTA DNA using LiCl/EtOH Precipitation
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Determination of the Fraction of Viable Cells with a Cell Sample

  Materials
  • Cell sample (105 to 107 cells/ml PBS; freshly isolated)
  • Trypan blue counting kit (Bio‐Rad, cat. no. 145‐0003)
  • Plasticware (e.g., reaction tubes, pipet tips, etc.) taken freshly from a new package
  • Automated cell counter TC20 (Bio‐Rad, cat. no. 145‐0102)
NOTE: Do not use autoclaved plastic material. Plastic material can be contaminated by DNA during the autoclaving process.NOTE: Use nitrile gloves for all steps.

Basic Protocol 2: Preparation of Cell Samples by Dilution

  Materials
  • Cell suspension of viable cells as determined in protocol 1
  • Phosphate‐buffered saline (PBS; iso‐osmotic cell dilution buffer, freshly prepared)
  • Plasticware (e.g., reaction tubes, pipet tips, etc.) taken freshly from a new package
NOTE: Use nitrile gloves for all steps.NOTE: Do not use autoclaved plastic material or plastic material from an open box already used for other laboratory work.

Basic Protocol 3: LYSE Cell Samples

  Materials
  • Cell sample with appropriate cell number ( protocol 2)
  • REPLI‐g WTA Single Cell Kit (Qiagen, cat. no. 150063 or 150065) containing the lysis buffer
  • Centrifuge
  • Plasticware (e.g., reaction tubes, pipet tips, etc.) taken from a new package.
  • Thermal cycler or water bath adjustable to various temperatures
NOTE: Do not use autoclaved plastic material or plastic material from an open box already used for other laboratory work.

Alternate Protocol 1: Use of Purified RNA Samples Instead of Cells for Whole‐Transcriptome Amplification

  Materials
  • Purified RNA
  • REPLI‐g Cell WTA Single Cell Kit (Qiagen, cat. no. 150063 or 150065) containing the NA denaturation buffer
  • Plasticware (e.g., reaction tubes, pipet tips, etc.) taken from a new package.
  • Centrifuge
  • Thermal cycler or water bath(s) adjustable to various temperatures
NOTE: In order to avoid contaminations by foreign DNA or RNA, do not use autoclaved plastic material or plastic material from an open box already used for other laboratory work.

Basic Protocol 4: Whole Transcriptome Amplification (WTA)

  Materials
  • 11 μl aliquot of lysate for WTA ( protocol 3) or of purified nucleic acid sample ( protocol 4Alternate Protocol)
  • REPLI‐g WTA Single Cell Kit (Qiagen, cat. no. 150063 or 150065) containing:
    • RT/polymerase buffer
    • gDNA wipeout buffer
    • H 2O sc
    • Random primer
    • Oligo dT primer
    • Quantiscript RT enzyme mix
    • Ligase buffer
    • Ligase mix
    • REPLI‐g sc reaction buffer
    • REPLI‐g SensiPhi DNA polymerase
  • Plasticware (e.g., reaction tubes, pipette tips, etc.) taken from a new package
  • Thermal cycler or water bath(s) adjustable to various temperatures
NOTE: Do not use autoclaved plastic material or plastic material from an open box already used for other laboratory work.

Basic Protocol 5: Use of WTA DNA for Next‐Generation Sequencing

  Materials
  • WTA DNA (see protocol 5)
  • Quant‐iT PicoGreen dsDNA reagent (Invitrogen, cat no. P7581) or Qubit dsDNA BR Assay system (Invitrogen, cat. no. Q32850)
  • TE buffer
  • MinElute PCR Purification kit (Qiagen, cat. no. 28004 or 28006)
  • Agilent DNA Chip 7500 (Agilent, cat. no. 5067‐150).
  • GeneRead NGS Library I Core kit (Qiagen, cat. no. 180432)
  • GeneRead Adaptor I kit (Qiagen, cat. no. 180912 or 180984)
  • GeneRead Library Quant Kit (Qiagen, cat. no. 180612) or Real‐time PCR Kapa Kit (peqLab, cat. no. 07‐KK4822).
  • 1.5‐ml microcentrifuge tubes

Support Protocol 1: Quality Control of WTA DNA using Real‐Time PCR

  Materials
  • WTA DNA (see protocol 6)
  • TE buffer (10 mM Tris·Cl; 1 mM EDTA, pH 8.0)
  • Agencourt AMPure XP beads (Beckman Coulter, cat. no. A63880)
  • 70% (v/v) ethanol (stored for no longer than 7 days)
  • Plasticware (e.g., reaction tubes, pipet tips, etc.) taken from a new package
  • Magnetic Particle Concentrator (e.g., Dyna Mag; Life Technologies, cat. no. 123.21)
  • 1.5‐ or 2‐ml microcentrifuge tubes
NOTE: Use nitrile gloves for all steps.NOTE: Use the ethanol and TE exclusively for WTA DNA.

Support Protocol 2: Optional Cleanup of WTA DNA using Agencourt Ampure XP Magnetic Beads

  Materials
  • WTA DNA (see protocol 6)
  • TE buffer (10 mM Tris·Cl; 1 mM EDTA, pH 8.0)
  • Lithium chloride (7.5 M)
  • EDTA (0.5 M, pH 8.0)
  • Ethanol (96% to 100%)
  • Ethanol (70%)
  • 1.5‐ or 2‐ml microcentrifuge tubes
  • Plasticware (e.g., reaction tubes, pipet tips, etc.) taken from a new package
  • Microcentrifuge
  • 45°C incubator
NOTE: Use nitrile gloves for all steps.NOTE: Use the ethanol and TE exclusively for WGA DNA.
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Figures

Videos

Literature Cited

Literature Cited
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