Isolation of Nascent Transcripts with Click Chemistry

Ozlem Yildirim1

1 Department of Genetics, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.24
DOI:  10.1002/0471142727.mb0424s111
Online Posting Date:  July, 2015
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Abstract

Steady‐state levels of cellular RNA are determined by both transcriptional rate and RNA half‐life. Commonly used methods for transcriptional analysis are only capable of profiling total RNA and do not distinguish changes in synthesis and decay rates. Hence, a better understanding of the temporal dynamics of cellular response for a given condition at the transcriptional level requires techniques for the analysis of nascent transcripts. Here we describe a protocol that allows isolation of nascent transcripts with a copper‐catalyzed azide‐alkyne cycloaddition (CuAAC) also known as a click chemistry reaction. © 2015 by John Wiley & Sons, Inc.

Keywords: nascent RNA; metabolic labeling; click chemistry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Cell‐Cycle‐Specific Nascent Transcripts with Click Chemistry
  • Support Protocol 1: Assessing Click Efficiency with RNA Dot‐Blot
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Cell‐Cycle‐Specific Nascent Transcripts with Click Chemistry

  Materials
  • Cultured cells (e.g., hTERT immortalized RPE1 cells; 2 × 106 to 5 × 106 cells for each experiment)
  • RPE1 medium (see recipe)
  • 5‐Ethynyl uridine (Berry & Associates, cat. no. PY7563)
  • TRIzol reagent (Life Technologies, cat. no. 15596‐026)
  • Nuclease‐free water (Ambion, cat. no. AM9932)
  • Chloroform (Sigma‐Aldrich, cat. no. C2432)
  • 100% and 80% ethanol
  • Isopropanol (Sigma‐Aldrich, cat. no. I9516)
  • 10× Baseline‐ZERO reaction buffer
  • Baseline‐ZERO DNase (Epicentre, cat. no. DB0715K)
  • RNA Clean & Concentrator kit (Zymo Research, cat. no. R1015)
  • Acetonitrile (Sigma‐Aldrich, cat. no. 34998)
  • 1 M Tris·HCl, pH 7.5
  • Sodium‐L‐ascorbate (Sigma‐Aldrich)
  • Copper sulfate (CuSO 4; Sigma‐Aldrich, cat. no. 451657)
  • Biotin‐dPEG 7‐azide (Quanta BioDesign, cat. no. 10825)
  • N,N,N′,N′′,N′′‐pentamethyldiethylenetriamine (PMDTA; Sigma‐Aldrich, cat. no. 369497)
  • L‐Mimosine (Sigma‐Aldrich, cat. no. M0253)
  • Glycogen (Ambion, cat. no. AM9510)
  • Dynabeads MyOne Streptavidin C1 magnetic beads (Life Technologies, cat. no. 65001)
  • 3 M sodium acetate, pH 5.5 (Ambion, cat. no. AM9740)
  • RNasin Plus Rnase inhibitor (Promega)
  • Ribo‐Zero Gold Magnetic kit (Epicentre, cat. no. MRZG12324)
  • SuperScript VILO cDNA synthesis kit (Life Technologies, cat. no. 11754‐050)
  • RNAse‐free water
  • 0.5 M EDTA
  • 1 N NaOH
  • 1 M HEPES
  • 100 mM DTT (Life Technologies, cat. no. 18080‐044)
  • SuperScript III reverse transcriptase (Life Technologies, cat. no. 18080‐044)
  • Random primers (Life Technologies, cat. no. 48190011)
  • 10 mM dNTPs
  • First Strand buffer (Life Technologies, cat. no. 18080‐044)
  • 5× Second Strand buffer (Life Technologies, cat. no. 10812‐014)
  • E. coli DNA ligase (New England BioLabs, cat. no. M0205S)
  • E. coli DNA polymerase (New England BioLabs, cat. no. M0209L)
  • E. coli RNase H (New England BioLabs, cat. no. M0297S)
  • Uracil DNA glycosylase (UDG; New England BioLabs, cat. no. M0280S)
  • TNE bead‐blocking buffer (see recipe)
  • TNE2.0 (see recipe)
  • TNE0.2 (see recipe)
  • Wash buffer 65 (see recipe)
  • RNaseZap (Ambion, cat. no. AM9780)
  • Amicon YM‐10 Ultra‐0.5 Centrifugal Filter Unit (EMD Millipore, cat. no. UFC5010BK)
  • Ampure XP beads (Beckman Coulter Genomics, cat. no. A63881)
  • Dyna Mag magnetic stand (Life Technologies, cat. no. 12321D)
  • Cell lifter (Corning, cat. no. 3008)
  • Low‐binding, nuclease‐free 1.7‐ml tubes (Eppendorf, cat. no. 022431021)
  • Thermomixer (Eppendorf, cat. no. 022670107)
  • Rotator (Thomas Scientific, cat. no. 1217H25)
  • Thermocycler
NOTE: All solutions must be prepared with Rnase‐free water.

Support Protocol 1: Assessing Click Efficiency with RNA Dot‐Blot

  Additional Materials (also see protocol 1Basic Protocol)
  • Formamide (Sigma‐Aldrich, cat. no. F9037)
  • 20× SSC (Ambion, cat. no. AM9763)
  • Pierce High Sensitivity Streptavidin‐HRP (Life Technologies, cat. no. 21134)
  • Nucleic Acid Detection blocking buffer (Thermo Scientific, cat. no. 89880 A)
  • Binding solution (see recipe)
  • Streptavidin‐HRP wash buffer (see recipe)
  • Zeta‐Probe membrane (Bio‐Rad, cat. no. 162‐0153)
  • Diethyl pyrocarbonate (DEPC)‐treated water (see recipe)
  • Tris‐buffered saline and Tween 20 (TBST)
  • Nuclease‐free BSA (Sigma‐Aldrich, cat. no. B2518)
  • Whatman 3 MM paper
  • Bio‐Dot apparatus (Bio‐Rad)
  • UV crosslinker (UV Stratalinker 2400)
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Figures

Videos

Literature Cited

Literature Cited
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