Investigation of Viral and Host Chromatin by ChIP‐PCR or ChIP‐Seq Analysis

Thomas Günther1, Juliane M. Theiss2, Nicole Fischer2, Adam Grundhoff1

1 Heinrich‐Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, 2 Institute for Medical Microbiology, Virology and Hygiene; University Medical Center Hamburg‐Eppendorf, Hamburg
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 1E.10
DOI:  10.1002/9780471729259.mc01e10s40
Online Posting Date:  February, 2016
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Complex regulation of viral transcription patterns and DNA replication levels is a feature of many DNA viruses. This is especially true for those viruses which establish latent or persistent infections (e.g., herpesviruses, papillomaviruses, polyomaviruses, or adenovirus), as long‐term persistence often requires adaptation of gene expression programs and/or replication levels to the cellular milieu. A key factor in the control of such processes is the establishment of a specific chromatin state on promoters or replication origins, which in turn will determine whether or not the underlying DNA is accessible for other factors that mediate downstream processes. Chromatin immunoprecipitation (ChIP) is a powerful technique to investigate viral chromatin, in particular to study binding patterns of modified histones, transcription factors or other DNA‐/chromatin‐binding proteins that regulate the viral lifecycle. Here, we provide protocols that are suitable for performing ChIP‐PCR and ChIP‐Seq studies on chromatin of large and small viral genomes.© 2016 by John Wiley & Sons, Inc.

Keywords: chromatin; ChIP; ChIP‐PCR; ChIP‐seq; viral chromatin; next‐generation sequencing

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Table of Contents

  • Introduction
  • Basic Protocol 1: Basic Chromatin Immunoprecipitation Protocol
  • Support Protocol 1: Evaluation of Shearing Efficiency by Agarose Gel Electrophoresis and Southern Blot Analysis
  • Basic Protocol 2: Quantification of Enriched Dna by qPCR
  • Basic Protocol 3: Identification of Enriched DNA by NGS Analysis (ChIP‐Seq)
  • Reagents And Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Basic Chromatin Immunoprecipitation Protocol

  • Cells expressing your target factor and harboring your target factor binding site; if applicable, use appropriate negative control cells (see Critical Parameters and Troubleshooting for appropriate samples and controls)
  • Cell culture medium
  • Trypsin
  • Formaldehyde (37%)
  • 1.25 M glycine in H 2O
  • Phosphate‐buffered saline (PBS; Sigma‐Aldrich, cat. no. D8537)
  • Protease inhibitor cocktail tablets (cOmplete; Roche, cat. no. 11697498001)
  • 1 M PMSF stock solution in dimethyl sulfoxide (DMSO)
  • Buffer I (see recipe)
  • Buffer II (see recipe)
  • Buffer III (see recipe)
  • Ice
  • 10% Triton X‐100
  • Dilution buffer (see recipe)
  • Protein G Sepharose Beads (4 fast flow, GE Healthcare, cat. no. 17‐0618‐01) or agarose‐A/G beads depending on antibody subtype (alternatively, magnetic beads and magnetic stand separation can be used)
  • BSA fraction V (e.g., NEB, cat. no. B9000S)
  • Antibodies against histone modifications or transcription factors: antibodies should be of high quality, preferably ChIP grade; examples for antibodies that worked well in our hands:
  • α‐LT: CM2B4 mouse monoclonal antibody (Santa Cruz, Biotechnology, cat. no. sc‐136172)
  • α‐H3K4‐me3: rabbit monoclonal, clone MC315 (Merck Millipore, cat. no. 04‐745)
  • Normal mouse or rabbit IgG (same species as your ChIP antibody, Merck Millipore, cat. no. 12‐370 or 12‐371)
  • Low‐salt wash buffer (see recipe)
  • High‐salt wash buffer (see recipe)
  • LiCl wash buffer (see recipe)
  • TE buffer (see recipe)
  • Elution buffer (see recipe)
  • 5 M NaCl
  • RNase A (10 mg/ml, Life Technologies, cat. no. 12091‐039)
  • CaCl 2 stock solution (see recipe)
  • Proteinase K (20 mg/ml; Peqlab, cat. no. 04‐1075)
  • Phenol/Chloroform/Isoamyl alcohol (25:24:1)
  • Chloroform
  • Glycogen (5 mg/ml; Life Technologies, cat. no. AM9510)
  • Absolute ethanol
  • Ethanol (70%)
  • 10 mM Tris·Cl, pH 8.0 (APPENDIX appendix 2A)
  • 10‐cm cell culture dishes for adherent cells
  • 37°C, 5% CO 2 humidified incubator
  • 15‐, 1.5‐, and 2‐ml reaction tubes
  • Centrifuge (4°C) for 1.5‐ml and 15‐ml tubes
  • Rotating wheel at 4°C
  • 1‐ml pipets
  • Sonication device for shearing of nucleic acids (Bioruptor, Diagenode)
  • Vortex mixer
  • Sterile blade, optional
  • Shaking heat block (37°C, 55°C, 65°C)
  • 2‐ml PhaseLock heavy tubes (5 prime, cat. no. 2302830)
  • Vacuum centrifuge or heat block
IMPORTANT NOTE: For ChIP‐Seq, beads must not be blocked with salmon sperm DNA or any kind of DNA since all DNA will be sequenced.

Support Protocol 1: Evaluation of Shearing Efficiency by Agarose Gel Electrophoresis and Southern Blot Analysis

  • Input sample DNA (see protocol 1)
  • DNA loading buffer: Bromphenol blue may be added to the loading buffer for the DNA size marker, but should not be used for chromatin samples (see step annotations)
  • 1.5% agarose gel
  • RedSafe Nucleic Acid Staining Solution (Bulldog Bio, cat. no. 21141)
  • DNA size marker (range 100 bp to 10,000 bp)
  • P32‐ labeled oligonucleotide probes
  • Standard electrophoresis equipment
  • UV gel documentation device
  • Nylon membrane (GE Healthcare, Amersham Hybond‐N+)
  • Standard Southern blot equipment
  • Additional reagents and equipment for Southern blot analysis (see Southern, and Brown, )
NOTE: The input DNA used in this protocol has been treated with RNAseA, which is the standard procedure within protocol 1. If no RNase A treatment has been performed, tRNAs may give a strong signal at the expected size of the sheared DNA.

Basic Protocol 2: Quantification of Enriched Dna by qPCR

  • Input DNA, ChIP DNA and optionally IgG control ChIP DNA (from protocol 1)
  • Primers for amplification of 70 to 120 nt of the target factor binding sites and negative control sites
  • 2× SYBR Green Mastermix (SensiMix; Bioline, cat. no. QT605‐05)
  • Ice
  • Rotor‐Gene Q (Qiagen)

Basic Protocol 3: Identification of Enriched DNA by NGS Analysis (ChIP‐Seq)

  • NEXTflex ChIP‐Seq Kit (Bioo Scientific, cat. nos. 5143‐01 and 5143‐02)
  • Qubit HS dsDNA kit (Life Technologies, cat. no. Q32851)
  • 0.1% Tween‐20
  • Bioanalyzer High sensitivity DNA kit (Agilent, cat. no. 5067‐4626)
  • 2100 Bioanalyzer instrument
  • Qubit 2.0 Fluorometer (Life Technologies)
  • Qubit assay tubes (Life Technologies, cat. no. Q32856)
  • Low‐binding tubes (Eppendorf, Lobind tubes 1.5 ml DNA/RNA, cat. no. 666548‐250)
  • Illumina HiSeq instrument
  • Linux‐based workstation
  • Bowtie (Langmead et al., )
  • MACS14 (Zhang et al., )
NOTE: For additional reagents and equipment for the NEXTflex ChIP‐Seq library preparation refer to the manufacturer's protocol.
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