Single‐Assay Profiling of Nucleosome Occupancy and Chromatin Accessibility

April Cook1, Jakub Mieczkowski2, Michael Y. Tolstorukov3

1 Current address: Dana‐Farber Cancer Institute, Boston, Massachusetts, 2 Current address: Neurobiology Center, Nencki Institute of Experimental Biology, Warsaw, 3 Department of Molecular Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.34
DOI:  10.1002/cpmb.45
Online Posting Date:  October, 2017
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Abstract

This unit describes a method for determining the accessibility of chromatinized DNA and nucleosome occupancy in the same assay. Enzymatic digestion of chromatin using micrococcal nuclease (MNase) is optimized for liberation, retrieval, and characterization of DNA fragments from chromatin. MNase digestion is performed in a titration series, and the DNA fragments are isolated and sequenced for each individual digest independently. These sequenced fragments are then collectively analyzed in a novel bioinformatics pipeline to produce a metric describing MNase accessibility of chromatin (MACC) and nucleosome occupancy. This approach allows profiling of the entire genome including regions of open and closed chromatin. Moreover, the MACC protocol can be supplemented with a histone immunoprecipitation step to estimate and compare both histone and non‐histone DNA protection components. © 2017 by John Wiley & Sons, Inc.

Keywords: chromatin; nucleosomes; accessibility; MNase titration

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: MNase Digestion of Chromatin and Purification of Protected DNA For Library Construction
  • Support Protocol 1: Buffer A+ Concentration Determination for MACC Assay
  • Support Protocol 2: Optimization of MNase Concentration Range
  • Basic Protocol 2: Data Processing and Generation of MNase Accessibility and Nucleosome Occupancy Profiles
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: MNase Digestion of Chromatin and Purification of Protected DNA For Library Construction

  Materials
  • 16% (w/v) formaldehyde
  • Cells of interest, in suspension or adherent
  • Cell culture medium appropriate for cells of interest
  • Glycine
  • Phosphate‐buffered saline (PBS; see appendix 22)
  • Buffers A, A+, and W (see recipe and Support Protocols protocol 21 and protocol 32)
  • 0.1 M CaCl 2
  • MNase (U/ml determined per lot; see protocol 3)
  • 0.5 M EDTA
  • 0.5 M EGTA
  • 10% (w/v) SDS
  • LB3 (see recipe)
  • Histone H3 and H4 antibodies (e.g., Abcam, cat. no. ab1791 and ab10158, respectively)
  • Dynabeads protein A
  • Elution buffer (see recipe)
  • 1 M Tris·Cl, pH 8 (see appendix 22)
  • 0.1 mg/µl RNase solution (e.g., Qiagen)
  • SDS buffer (see recipe)
  • 20 µg/µl proteinase K (e.g., Roche, cat. no. 03115828001)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (PCI; see unit 2.1; Moore & Dowhan, )
  • 15‐ml phase lock gel‐containing tube
  • 3 M sodium acetate
  • 100% ethanol
  • 20 µg/µl glycogen
  • Tris/EDTA (TE) buffer (see appendix 22)
  • Laboratory shaker
  • Centrifuge and microcentrifuge
  • Liquid nitrogen
  • 15‐ml Dounce tissue grinder with pestle
  • Hemocytometer
  • Microscope suitable for hemocytometer use
  • 50‐ml conical tubes
  • 1.5‐ml low‐retention microcentrifuge tubes
  • Vortex
  • Rotating mixer or tube rotator (with end‐over‐end capabilities)
  • Incubator or heating block, with variable temperature
  • Dry ice
  • Agilent Bioanalyzer
  • Additional reagents and equipment for immunoprecipitation (see unit 21.3), PCI DNA extraction (see unit 2.1; Moore & Dowhan, ), and high‐throughput sequencing (see Bowman et al., )
NOTE: Reagents should be prepared in sterile, disposable labware, taken directly from its packaging, or in glassware that has been soaked in 10% bleach, thoroughly rinsed in tap water followed by distilled water, and autoclaved. When applicable, reagents should be filter‐ or autoclave‐sterilized. MNase in solution should be stored in aliquots at −80°C. Formaldehyde should be freshly prepared, as should sucrose‐, DTT‐, and protease inhibitor−containing solutions.

Support Protocol 1: Buffer A+ Concentration Determination for MACC Assay

  Materials
  • Reagents and equipment for nuclei extraction, MNase digestion, DNA isolation, and assessment of degrees of digestion (see protocol 1)

Support Protocol 2: Optimization of MNase Concentration Range

  Materials
  • A computer running Linux, Windows, or Mac OS X
  • R environment (version 3.0 and higher; available at https://www.r‐project.org/) and installed R packages: “parallel,” “descr,” “limma,” “GenomicRanges,” “Rsamtools,” and “MACC” available at http://genetics.mgh.harvard.edu/MACC
  • Input files providing information on the genomic locations of sequenced reads
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Figures

Videos

Literature Cited

Literature Cited
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