Transcriptome Analysis at the Single‐Cell Level Using SMART Technology

Rachel N. Fish1, Magnolia Bostick1, Alisa Lehman2, Andrew Farmer1

1 Takara Bio USA, Inc. (formerly Clontech Laboratories, Inc.), Mountain View, 2 Current affiliation: 23andMe, Mountain View
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.26
DOI:  10.1002/cpmb.23
Online Posting Date:  October, 2016
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Abstract

RNA sequencing (RNA‐seq) is a powerful method for analyzing cell state, with minimal bias, and has broad applications within the biological sciences. However, transcriptome analysis of seemingly homogenous cell populations may in fact overlook significant heterogeneity that can be uncovered at the single‐cell level. The ultra‐low amount of RNA contained in a single cell requires extraordinarily sensitive and reproducible transcriptome analysis methods. As next‐generation sequencing (NGS) technologies mature, transcriptome profiling by RNA‐seq is increasingly being used to decipher the molecular signature of individual cells. This unit describes an ultra‐sensitive and reproducible protocol to generate cDNA and sequencing libraries directly from single cells or RNA inputs ranging from 10 pg to 10 ng. Important considerations for working with minute RNA inputs are given. © 2016 by John Wiley & Sons, Inc.

Keywords: RNA‐seq; transcriptome; gene expression; next‐generation sequencing; single cell

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: cDNA Synthesis for NGS
  • Basic Protocol 2: Illumina Library Preparation Using Tagmentation
  • Alternate Protocol 1: Illumina Library Preparation Using Covaris Shearing and Ligation‐Based Adapter Addition
  • Alternate Protocol 2: Library Preparation Using Ion Torrent Sequencing Platforms
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: cDNA Synthesis for NGS

  Materials
  • SMART‐Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech, cat. no. 634888‐634894) containing:
  • 10× Lysis Buffer
  • RNase Inhibitor (40 U/µl)
  • Nuclease‐Free Water
  • 3′ SMART‐Seq CDS Primer II A (12 µM)
  • 5× Ultra Low First‐Strand Buffer
  • SMART‐Seq v4 Oligonucleotide (48 µM)
  • SMARTScribe Reverse Transcriptase (100 U/µl)
  • SeqAmp PCR Buffer
  • PCR Primer II A (12 µM)
  • SeqAmp DNA Polymerase
  • Elution Buffer (10 mM Tris⋅Cl, pH 8.5)
  • Total RNA or cells in PBS
  • Agencourt AMPure XP PCR purification kit (5 ml, Beckman Coulter, cat. no. A63880; 60 ml, Beckman Coulter, cat. no. A63881)
  • 80% (v/v) ethanol, freshly prepared using nuclease‐free H 2O
  • High Sensitivity DNA Kit (Agilent, cat. no. 5067‐4626)
  • Nuclease‐free thin‐wall 0.2‐ml PCR tubes or 8‐well strips (USA Scientific, cat. no. 1402‐4700)
  • Hot‐lid thermal cycler
  • Magnetic separation device for small volumes
  • Nuclease‐free low‐adhesion 1.5‐ml tubes (USA Scientific, cat. no. 1415‐2600) or LoBind tubes (Eppendorf, cat. no. 022431021)
  • Agilent 2100 Bioanalyzer

Basic Protocol 2: Illumina Library Preparation Using Tagmentation

  Materials
  • Nextera XT DNA Library Preparation Kits (Illumina, cat. nos. FC‐131‐1024 and FC‐131‐1096) containing:
  • TD (Tagment DNA Buffer)
  • ATM (Amplicon Tagment Mix)
  • NT (Neutralize Tagment Buffer)
  • NPM (Nextera PCR Master Mix)
  • Index 1 primers (N7XX)
  • Index 2 primers (S5XX)
  • RSB (Resuspension Buffer)
  • LNA1 (Library Normalization Additives 1)
  • LNB1 (Library Normalization Beads 1)
  • LNW1 (Library Normalization Wash 1)
  • LNS1 (Library Normalization Storage Buffer 1)
  • Purified cDNA (100 to 150 pg; protocol 1)
  • Agencourt AMPure XP PCR purification kit (5 ml, Beckman Coulter, cat. no. A63880; 60 ml, Beckman Coulter, cat. no. A63881)
  • 80% (v/v) ethanol, freshly prepared using nuclease‐free H 2O
  • High Sensitivity DNA Kit (Agilent, cat. no. 5067‐4626)
  • 0.1 N NaOH prepared with nuclease‐free H 2O
  • 0.2‐ml PCR tubes or 96‐well PCR plate
  • Heated‐lid thermal cycler
  • Magnetic separation device
  • Nuclease‐free low‐adhesion 1.5‐ml tubes (USA Scientific, cat. no. 1415‐2600) or LoBind tubes (Eppendorf, cat. no. 022431021)
  • Agilent 2100 Bioanalyzer
  • 96‐well plates compatible with magnetic separation device used (e.g., Fisher Scientific, cat. no. AB‐0859 or Axygen, cat. no. P‐96‐450V‐C‐S)
  • Adhesive plate sealers
  • Microplate shaker

Alternate Protocol 1: Illumina Library Preparation Using Covaris Shearing and Ligation‐Based Adapter Addition

  Materials
  • Purified cDNA (50 pg to 20 ng; protocol 1)
  • Elution Buffer (from the SMART‐Seq v4 Ultra Low Input RNA Kit for Sequencing)
  • Low Input Library Prep Kit v2 or HT (Clontech, cat. no. 634899 or 634900) containing:
  • Template Preparation Buffer
  • Template Preparation Enzyme
  • Library Synthesis Buffer
  • Library Synthesis Enzyme
  • Nuclease‐Free Water
  • Library Amplification Buffer
  • Library Amplification Enzyme
  • Indexing Reagents 1 to 12 or Dual Index Plate (96D)
  • Agencourt AMPure XP PCR purification kit (5 ml, Beckman Coulter, cat. no. A63880; 60 ml, Beckman Coulter, cat. no. A63881)
  • 80% (v/v) ethanol, freshly prepared using nuclease‐free H 2O
  • Covaris AFA system (S220 Focused‐ultrasonicator)
  • 100‐µl Covaris microTUBE AFA Fiber Pre‐Slit Snap‐Cap (Covaris, cat. no. 520045)
  • Covaris Sample holder (S holder 500114)
  • Nuclease‐free 0.2‐ml thin wall PCR tubes with caps
  • Heated‐lid thermal cycler
  • 96‐well Axygen V‐bottom plate (VWR, cat. no. 47743‐996)
  • MicroAmp Clear Adhesive Film (Thermo Fisher Scientific, cat. no. 4306311)
  • Magnetic Stand‐96 (Thermo Fisher Scientific, cat. no. AM10027)
  • Nuclease‐free low‐adhesion 1.5‐ml tubes (USA Scientific, cat. no. 1415‐2600) or LoBind tubes (Eppendorf, cat. no. 022431021)

Alternate Protocol 2: Library Preparation Using Ion Torrent Sequencing Platforms

  Materials
  • Ion Xpress Plus Fragment Library Kit (Thermo Fisher Scientific, cat. no. 4471269) containing:
  • Ion Shear Plus 10× Reaction Buffer
  • Ion Shear Plus Enzyme Mix II
  • Ion Shear Plus Stop Buffer
  • Purified cDNA (1 to 10 ng; protocol 1)
  • Nuclease‐free H 2OAfaI (10 U/µl; Clontech, included in cat. nos. 634888 to 634894)
  • Nuclease‐free low‐adhesion 1.5‐ml tubes (USA Scientific, cat. no. 1415‐2600) or LoBind tubes (Eppendorf, cat. no. 022431021)
  • Heated‐lid thermal cycler
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Figures

Videos

Literature Cited

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