Genome‐Scale Mapping of DNase I Hypersensitivity

Sam John1, Peter J. Sabo1, Theresa K. Canfield2, Kristen Lee2, Shinny Vong2, Molly Weaver2, Hao Wang2, Jeff Vierstra2, Alex P. Reynolds2, Robert E. Thurman2, John A. Stamatoyannopoulos3

1 Authors contributed equally to this work., 2 Department of Genome Sciences, University of Washington, Seattle, Washington, 3 Department of Medicine, University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.27
DOI:  10.1002/0471142727.mb2127s103
Online Posting Date:  July, 2013
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Abstract

DNase I‐seq is a global and high‐resolution method that uses the nonspecific endonuclease DNase I to map chromatin accessibility. These accessible regions, designated as DNase I hypersensitive sites (DHSs), define the regulatory features, (e.g., promoters, enhancers, insulators, and locus control regions) of complex genomes. In this unit, methods are described for nuclei isolation, digestion of nuclei with limiting concentrations of DNase I, and the biochemical fractionation of DNase I hypersensitive sites in preparation for high‐throughput sequencing. DNase I‐seq is an unbiased and robust method that is not predicated on an a priori understanding of regulatory patterns or chromatin features. Curr. Protoc. Mol. Biol. 103:21.27.1–21.27.20. © 2013 by John Wiley & Sons, Inc.

Keywords: chromatin; nucleosome; DNase I‐seq; transcription; regulatory DNA

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Nuclei and Digestion with DNase I
  • Basic Protocol 2: Biochemical Fractionation of DNase I Hypersensitive Sites
  • Basic Protocol 3: Preparation of DHS Libraries for Sequencing on the Illumina Genome Analyzer
  • Basic Protocol 4: Quality Control for DNase I Digestion
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Nuclei and Digestion with DNase I

  Materials
  • Complete EDTA‐free Protease Inhibitor tablets (Roche Applied Science, cat. no. 04‐693‐132‐001)
  • Buffer A (see recipe)
  • Ice
  • 2 × IGEPAL CA‐630 solution (see recipe)
  • Spermidine‐free base (see recipe)
  • Spermine‐free base (see recipe)
  • Stop buffer (see recipe)
  • 10× DNase I digestion buffer (see recipe)
  • 25 to 50 million mammalian cells
  • Phosphate‐buffered saline (Cellgro, cat. no. 21‐040‐CM)
  • Proteinase K (Sigma‐Aldrich, cat. no. P4850)
  • Ribonuclease A (RNase A; Sigma‐Aldrich, cat.no. R4642)
  • 20‐, 200‐, and 1000‐µl Pipetman (Rainin, cat. nos. L‐20, L‐200, and L‐100)
  • 20‐, 200‐, and 1000‐µl pipet tips
  • Graduated pipets (5, 10, 25, 50 ml)
  • Digital 10‐liter water baths (Fisher Scientific, cat. no. 15‐462‐10Q)
  • 15‐ml polypropylene conical centrifuge tubes (Corning, cat. no. 430766)
  • Benchtop centrifuge (Eppendorf, model no. 5810R)
  • Swinging‐bucket rotor (Eppendorf, cat. no. A‐4‐62)
  • Phase hemacytometer (VWR, cat. no. 15170‐263) or an automatic cell counter
  • Inverted phase‐contrast microscope (Nikon, model no. TS‐100)

Basic Protocol 2: Biochemical Fractionation of DNase I Hypersensitive Sites

  Materials
  • Digested samples (see Basis Protocol 1)
  • Phenol‐Chloroform‐Isoamyl Alcohol (Invitrogen, cat.no. 15593‐031)
  • 9% sucrose gradient (see recipe)
  • 2% agarose gel (see unit 2.5)
  • SyBr Green I (Invitrogen, cat.no. S7563)
  • Qiagen MinElute Gel extraction kit (Qiagen, cat.no. 28604) containing:
    • QG buffer
    • MiniElute spin columns
    • Buffer PE (wash buffer)
  • 100% isopropanol
  • 15‐ml Phase Lock Heavy Tubes (5 Prime, cat.no. 2302850)
  • Benchtop centrifuge (Eppendorf, model no. 5810R)
  • Swinging‐bucket rotor (Eppendorf, cat.no. A‐4‐62)
  • 15‐ml Centricon, 10 kDa MWCO (EMD Millipore, cat.no. UFC 701008)
  • Open‐top thick‐walled polyallomer tubes (Seton Scientific, cat.no. 5032)
  • Standard laboratory ultracentrifuge (e.g.. Beckman LE‐80)
  • Electrophoresis equipment
  • Scanner (e.g., Typhoon 9200 scanner)
  • QIAvac 24 Plus Vacuum manifold (Qiagen, cat.no. 19413)
  • 10‐ml syringes
  • 1.7‐ml microcentrifuge tubes (Genesee Scientific, cat.no. 24‐282)
  • QIAvac 24 Plus Vacuum manifold (Qiagen, cat. no. 19413)
  • Fluorometer (e.g., Qubit, Invitrogen)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5)

Basic Protocol 3: Preparation of DHS Libraries for Sequencing on the Illumina Genome Analyzer

  Materials
  • Purified DNA (from protocol 2, step 14)
  • T4 DNA ligase (New England Biolabs, cat. no. M0202L) containing:
    • 10 × T4 DNA ligase buffer with ATP
  • 10 mM dNTP mix (Promega, cat. no. U1511)
  • T4 DNA polymerase
  • Klenow DNA polymerase (New England Biolabs, cat. No M0210L)
  • T4 polynucleotide kinase (PNK; New England Biolabs, cat. no. M0201S)
  • Qiagen MinElute Gel extraction kit (Qiagen, cat.no. 28604) containing:
    • Buffer PB
    • MinElute columns
    • Buffer EB
    • Buffer QG
  • Klenow (3′ to 5′ exo‐) (New England Biolabs, cat. no. M0212L)
  • 10× NEBuffer 2 (New England Biolabs, cat. no. B7002S)
  • NEB Quick Ligation kit (New England Biolabs, cat. no. M2200L) containing:
    • Quick ligation reaction buffer
    • Quick ligase
  • Illumina oligo adapter mix (Illumina, cat. no. PE‐102‐1004)
  • Kapa HiFi Library Amplification kit (KAPA Biosystems, KK2611) containing:
    • KAPA Hifi HotStart Taq polymerase
  • PCR primers 1.0 and 2.0 (standard Illumina primers)
  • Sterile Milli‐Q or molecular biology grade water
  • Loading dye
  • SyBr Green I
  • Preparative 2% agarose gel (1× TAE; see unit 2.5)
  • Isopropanol
  • Kapa Hifi Library Quantification kit (KAPA Biosystems, cat. no. KK4824)
  • 1.7‐ml microcentrifuge tubes (Genesee Scientific, cat. no. 24‐282)
  • 20° and 37°C incubators
  • Standard laboratory PCR machine (e.g., BioRad DNA Engine)
  • Electrophoresis equipment
  • Typhoon 9200 scanner
  • Vortex mixer
  • Illumina Genome Analyzer
  • Additional reagents and equipment for agarose gel electrophoresis (see unit 2.5)

Basic Protocol 4: Quality Control for DNase I Digestion

  Materials
  • DNase I‐digested DNA (see protocol 1, step 25)
  • 1% agarose gel
  • SyBr Green I in 1× TAE gel buffer
  • Qiagen MinElute PCR Purification kit (Qiagen, cat. no. 28004) containing:
    • Purification columns
  • q‐PCR reagents (e.g., Roche, cat. no. 04 710 436 001)
  • Electrophoresis apparatus
  • Typhoon 9200 scanner
  • Standard laboratory q‐PCR machine (e.g., ABI 7900HT)
  • Additional materials and equipment for quantitating DNA ( appendix 3J)
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Figures

Videos

Literature Cited

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