RAMPAGE: Promoter Activity Profiling by Paired‐End Sequencing of 5′‐Complete cDNAs

Philippe Batut1, Thomas R. Gingeras1

1 Cold Spring Harbor
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 25B.11
DOI:  10.1002/0471142727.mb25b11s104
Online Posting Date:  November, 2013
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Abstract

RNA annotation and mapping of promoters for analysis of gene expression (RAMPAGE) is a method that harnesses highly specific sequencing of 5′‐complete complementary DNAs to identify transcription start sites (TSSs) genome‐wide. Although TSS mapping has historically relied on detection of 5′‐complete cDNAs, current genome‐wide approaches typically have limited specificity and provide only scarce information regarding transcript structure. RAMPAGE allows for highly stringent selection of 5′‐complete molecules, thus allowing base‐resolution TSS identification with a high signal‐to‐noise ratio. Paired‐end sequencing of medium‐length cDNAs yields transcript structure information that is essential to interpreting the relationship of TSSs to annotated genes and transcripts. As opposed to standard RNA‐seq, RAMPAGE explicitly yields accurate and highly reproducible expression level estimates for individual promoters. Moreover, this approach offers a streamlined 2‐ to 3‐day protocol that is optimized for extensive sample multiplexing, and is therefore adapted for large‐scale projects. This method has been applied successfully to human and Drosophila samples, and in principle should be applicable to any eukaryotic system. Curr. Protoc. Mol. Biol. 104:25B.11.1‐25B.11.16. © 2013 by John Wiley & Sons, Inc.

Keywords: transcription start site; promoter; RAMPAGE; high‐throughput sequencing; expression profiling

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of 5′‐Complete cDNAs for Paired‐End Sequencing
  • Support Protocol 1: Preparation of tRNA Stock Solution
  • Basic Protocol 2: Analysis of Sequence Data Following Rampage
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of 5′‐Complete cDNAs for Paired‐End Sequencing

  Materials
  • DNaseI‐treated total RNA
  • Terminator (TEX) enzyme with buffer A (Epicentre, cat. no. TER51020)
  • Molecular‐biology grade water (Sigma‐Aldrich, cat. no. 95284‐100ML)
  • Agencourt RNAClean XP kit (Beckman Coulter, cat. no. A63987)
  • 70% (v/v) ethanol, freshly prepared
  • Reverse transcription (RT) primer:
    • 400 µM rampage_RT:
    • 5′‐TAGTCGAACGAAGGTCTCCGAACCGCTCTTCCGATCT(N) 15
  • Template‐switching oligonucleotides (TSOs, Table 25.11.1):
    • 4 mM rampage_TS_**:
    • 5′‐ TAGTCGAACTGAAGGTCTCCAGCANNNNNNrGrGrG
  • SuperScript III reverse transcriptase (Invitrogen, 200 U/µl, cat. no. 18080‐085), with first‐strand buffer and 100 mM DTT
  • 10 mM dNTP mix (Invitrogen, cat. no. 18427‐013)
  • Sorbitol/trehalose solution (see recipe)
  • 5 M betaine (Sigma‐Aldrich, cat. no. B0300‐1VL)
  • qPCR primers:
    • 10 µM CAGEscan‐erF:
    • 5′‐AATGATACGGCGACCACCGAGATCTACACTAGTCGAACTGAAGG
    • 10 µM CAGEscan‐erR:
    • 5′‐CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTG AACCGCTCTTCCGATCT
  • Power SYBR Green premix (Applied Biosystems, cat. no. 4367659)
  • Sodium periodate (NaIO 4, ≥99.8%, Sigma‐Aldrich, cat. no. 311448‐5G)
  • 1 M sodium acetate (NaOAc), pH 4.5: prepare from commercial 3 M NaOAc, pH 5.5 (Ambion, cat. no. AM9740)
  • 40% (v/v) glycerol (Sigma‐Aldrich, cat. no. G5516‐100ML)
  • 1 M Tris‐Cl, pH 7.0 and 8.5: prepare from commercial pH 7.4 stock (Sigma‐Aldrich, cat. no. T2194–100ML) by adjusting pH with HCl or NaOH
  • Biotin hydrazide, long arm (Vector Laboratories, cat. no. SP‐1100)
  • 1 M sodium citrate, pH 6.0 (Sigma‐Aldrich, cat. no. S1804‐500G)
  • 0.5 M EDTA, pH 8.0 (Ambion, cat. no. AM9260G)
  • 5 to 10 U/µl RNase I (Promega, cat. no. M4261)
  • 10 mg/ml MPG streptavidin beads (PureBiotech, cat. no. MSTR0502)
  • E. coli tRNA, DNA and protein free (see protocol 2Support Protocol)
  • Wash buffers 1 to 4 (see reciperecipes)
  • 10 M NaOH (Sigma‐Aldrich, cat. no. 72068‐100ML)
  • Agencourt AMPure XP kit (Beckman Coulter, cat. no. A63881)
  • Ex Taq Hot Start (HS) polymerase with buffer and 2.5 mM dNTP mix (Clontech, cat. no. RR006A)
  • Sequencing primers:
    • rampage_r1 (custom primer):
    • 5′‐ TAGTCGAACTGAAGGTCTCCAGCA
    • SBS8 (standard Illumina primer):
    • 5′‐ CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT

Support Protocol 1: Preparation of tRNA Stock Solution

  Materials
  • E. coli tRNA (type XX, Sigma‐Aldrich, cat. no. R1753‐500UN)
  • RQ1 RNase‐free DNase with buffer (Promega, cat. no. M6101)
  • 0.5 M EDTA, pH 8.0 (Ambion, cat. no. AM9260G)
  • 10% SDS (Sigma‐Aldrich, cat. no. G05030‐500ML‐F)
  • Proteinase K (New England Biolabs, cat. no. P8102S)
  • Agencourt RNAClean XP kit (Beckman Coulter, cat. no. A63987)
  • 70% (v/v) ethanol
  • 1.5‐ml microcentrifuge tube
  • Magnet for bead separation
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Figures

Videos

Literature Cited

Literature Cited
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