Single‐Assay Profiling of Nucleosome Occupancy and Chromatin Accessibility

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.

Low‐Input MNase Accessibility of Chromatin (Low‐Input MACC)

“…Cells of interest, either primary cells or cell lines (in suspension or adherent, see Support Protocol 1) 10% FBS/Ca 2+ /Mg 2+ -free PBS (or serum-free Ca 2+ /Mg 2+ -free PBS) 2× nuclei extraction buffer (see recipe) Protease inhibitor (e.g., cOmplete TM Protease Inhibitor Cocktail, Roche) MNase enzyme stock aliquots (see recipe) MNase digestion buffer (see recipe) 0.5 M EDTA 0.5 M EGTA RNase A (Thermo Fisher Scientific, cat #EN0531) 10% SDS Proteinase K (Ambion, cat #AM2548) Agencourt AMPure XP beads (e.g., from Beckman Coulter) Nuclease-free H 2 O 80% ethanol, ice cold Invitrogen Qubit R dsDNA HS Assay Kit (Life Technologies Corporation, cat #Q32851) Library preparation kit (e.g., NEBNext R Ultra TM II DNA Library Prep Kit for Illumina, cat #E7645S) 10 mM TrisCl, pH 8 (Moore, 1996) Hemocytometer or cell counting slides for automated cell counters Microscope suitable for hemocytometer use 1.5-ml low-retention microcentrifuge tubes 0.2-ml individual PCR tubes Thermal cycler Magnetic separator accommodating 0.2-ml tube Agilent Bioanalyzer or Agilent TapeStation with relevant reagents and accessories Illumina HiSeq 2500 sequencer Additional reagents and equipment for counting and handling of cells (Support Protocol 1), MNase dilution (Support Protocol 3), and data processing and generation of MACC metrics (Support Protocol 4 and Cook et al, 2017) NOTE: Reagents should be prepared in sterile, disposable labware or in glassware that has been soaked in 10% bleach, thoroughly rinsed in tap water followed by distilled water, and autoclaved. Reagents should be sterilized by filtration or autoclaving.…”

“…Data processing is described in Cook et al. (). A minimum of two biological replicates are required, with three being the ideal number.…”

“…The following protocol covers the enzymatic digestion of chromatin using MNase with low-cell-number input requirements. The protocol does not require crosslinking of samples with formaldehyde, as previously described for MACC (Cook et al, 2017;Mieczkowski et al, 2016). Comparison of MACC between formaldehyde-crosslinked and non-crosslinked cells was performed and no significant differences were detected.…”

“…Current Protocols in Molecular Biology 25. Process data and generate MACC metrics as previously described in Basic Protocol 2 of Single-Assay Profiling of Nucleosome Occupancy and Chromatin Accessibility (see Current Protocols article, Cook et al, 2017). Figure 2.…”

“…Recently, we developed a method for single‐assay profiling of nucleosome occupancy and chromatin accessibility that profiles both open and closed genomic loci simultaneously (Cook, Mieczkowski, & Tolstorukov, ; Mieczkowski et al., ). This method (called MACC, MNase accessibility) relies on a series of micrococcal nuclease (MNase) digestions of increasing depth to profile chromatin accessibility in a cell population.…”