Isolation of neuronal chromatin from brain tissue.

BMC neuroscience (2008-04-30)
Yan Jiang, Anouch Matevossian, Hsien-Sung Huang, Juerg Straubhaar, Schahram Akbarian
ABSTRACT

DNA-protein interactions in mature brain are increasingly recognized as key regulators for behavioral plasticity and neuronal dysfunction in chronic neuropsychiatric disease. However, chromatin assays typically lack single cell resolution, and therefore little is known about chromatin regulation of differentiated neuronal nuclei that reside in brain parenchyma intermingled with various types of non-neuronal cells. Here, we describe a protocol to selectively tag neuronal nuclei from adult brain - either by (anti-NeuN) immunolabeling or transgene-derived histone H2B-GFP fusion protein - for subsequent fluorescence-activated sorting and chromatin immunoprecipitation (ChIP). To illustrate an example, we compared histone H3 lysine 4 and 9 methylation marks at select gene promoters in neuronal, non-neuronal and unsorted chromatin from mouse forebrain and human cerebral cortex, and provide evidence for neuron-specific histone methylation signatures. With the modifications detailed in this protocol, the method can be used to collect nuclei from specific subtypes of neurons from any brain region for subsequent ChIP with native/un-fixed or crosslinked chromatin preparations. Starting with the harvest of brain tissue, ChIP-ready neuronal nuclei can be obtained within one day.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥98.5% (GC)
Sigma-Aldrich
IGEPAL® CA-630, viscous liquid
Sigma-Aldrich
Anti-NeuN Antibody, clone A60, clone A60, Chemicon®, from mouse
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, protease free, suitable for hybridization, pH 7, ≥98%
Sigma-Aldrich
Nuclease micrococcal from Staphylococcus aureus, 100-300 units/mg protein
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GenomePlex® Complete Whole Genome Amplification (WGA) Kit, Optimized kit with enzyme for amplifying a variety of DNA including FFPE tissue
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Atto 550, for fluorescence, ≥90% (HPCE)
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Anti-trimethyl-Histone H3 (Lys4) Antibody, Upstate®, from rabbit
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Atto 647, BioReagent, suitable for fluorescence, ≥90% (HPCE)
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Anti-dimethyl-Histone H3 (Lys4) Antibody, Upstate®, from rabbit
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Anti-trimethyl-Histone H3 (Lys9) Antibody, Upstate®, from rabbit
Sigma-Aldrich
Atto 647 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)
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Atto 550 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)
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Atto 550-Biotin, BioReagent, suitable for fluorescence, ≥90% (HPCE)
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Atto 647 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)
Sigma-Aldrich
Atto 550 azide, BioReagent, suitable for fluorescence, ≥80.0% (HPCE)
Sigma-Aldrich
Atto 550 amine, BioReagent, suitable for fluorescence

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