Isolating Viral and Host RNA Sequences from Archival Material and Production of cDNA Libraries for High‐Throughput DNA Sequencing

Yongli Xiao1, Zong‐Mei Sheng1, Jeffery K. Taubenberger1

1 Viral Pathogenesis and Evolution Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 1E.8
DOI:  10.1002/9780471729259.mc01e08s37
Online Posting Date:  May, 2015
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The vast majority of surgical biopsy and post‐mortem tissue samples are formalin‐fixed and paraffin‐embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. As an example, the viral RNA genome of the 1918 pandemic influenza A virus was previously determined in a 9‐year effort by overlapping RT‐PCR from post‐mortem samples. Using the protocols described here, the full genome of the 1918 virus was determined at high coverage in one high‐throughput sequencing run of a cDNA library derived from total RNA of a 1918 FFPE sample after duplex‐specific nuclease treatments. This basic methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of infectious diseases. © 2015 by John Wiley & Sons, Inc.

Keywords: RNA; cDNA; high‐throughput sequencing; library; influenza; polymerase chain reaction

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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of RNA from Archival Fixed Tissue Samples
  • Basic Protocol 2: Preparation of Sequencing Library
  • Basic Protocol 3: Normalization of Library Using Duplex‐Specific Thermostable Nuclease
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Isolation of RNA from Archival Fixed Tissue Samples

  • Tissue specimen in 6‐μm sections
  • CitriSolv (Fisher Scientific)
  • Absolute and 75% (v/v) ethanol
  • 20 mg/ml proteinase K in molecular‐biology‐grade water, stored at −20°C
  • Extraction buffer (see recipe)
  • TRIzol LS, stored at 4°C
  • Chloroform
  • 20 μg/μl glycogen, stored at −20°C
  • Isopropanol
  • DEPC‐treated water
  • 1 U/μl DNase I (amplification grade) and 10× DNase reaction buffer
  • 25 mM EDTA
  • Agilent RNA 6000 Nano kit (including RNA ladder, marker, gel matrix, dye concentrate, spin filter, and RNA chip)
  • 1.5‐ml RNase‐free microcentrifuge tubes
  • 0.5‐ml RNase‐free vials
  • 55°C oven
  • 55°, 65°, and 70°C water baths
  • Agilent 2100 Bioanalyzer (including IKA vortexer and adapter)

Basic Protocol 2: Preparation of Sequencing Library

  • Random primers
  • RNA from archival FFPE samples (see protocol 1)
  • DEPC‐treated water
  • SuperScript II
  • TruSeq RNA sample preparation kit v2 (including first and second strand master mixes, resuspension buffer, end repair mix, A‐tailing mix, ligation mix, RNA adapter index, stop ligation buffer, PCR primer cocktail, PCR master mix)
  • AMPure XP beads
  • 80% (v/v) ethanol
  • Agilent high‐sensitivity DNA kit (including gel matrix, dye concentrate, marker, ladder, spin filter, and high‐sensitivity DNA chip)
  • 0.2‐ml thin‐wall PCR tubes
  • Thermal cycler
  • 1.5‐ml microcentrifuge tubes
  • Magnetic stand
  • Agilent 2100 Bioanalyzer (including IKA vortexer and adapter)

Basic Protocol 3: Normalization of Library Using Duplex‐Specific Thermostable Nuclease

  • 1 M HEPES buffer, pH 7.2‐7.5
  • 5 M NaCl
  • DEPC‐treated water
  • Sequencing library (see protocol 2)
  • Duplex‐specific nuclease kit (Axxora; including 10× DSN master buffer, DSN enzyme, and 2× DSN stop solution)
  • AMPure XP beads
  • 80% (v/v) ethanol
  • EB buffer (Qiagen; 10 mM Tris·Cl, pH 8.5)
  • Phusion polymerase with 5× reaction buffer and 25 mM dNTPs (Life Technologies)
  • PCR primers PE 1.0 and 2.0 (Illumina)
  • 200‐μl PCR tubes, sterile and nuclease‐free
  • Thermal cycler
  • 68°C heat block
  • 1.5‐ml microcentrifuge tubes (if needed for magnetic stand)
  • Magnetic stand
  • Additional reagents and equipment for library validation (see protocol 2)
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Literature Cited

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