Abstract
In recent years, the assay for transposase-accessible chromatin using sequencing (ATAC-Seq) has become a fundamental tool of epigenomic research. However, it is difficult to perform this technique on frozen samples because freezing cells before extracting nuclei can impair nuclear integrity and alter chromatin structure, especially in fragile cells such as neurons. Our aim was to develop a protocol for freezing neuronal cells that is compatible with ATAC-Seq; we focused on a disease-relevant cell type, namely motor neurons differentiated from induced pluripotent stem cells (iMNs) from a patient affected by spinal muscular atrophy. We found that while flash-frozen iMNs are not suitable for ATAC-Seq, the assay is successful with slow-cooled cryopreserved cells. Using this method, we were able to isolate high quality, intact nuclei, and we verified that epigenetic results from fresh and cryopreserved iMNs quantitatively agree.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Cell Differentiation
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Cell Nucleus / drug effects*
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Cell Nucleus / metabolism
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Cell Nucleus / ultrastructure
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Cell Survival / drug effects
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Chromatin / drug effects*
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Chromatin / metabolism
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Chromatin / ultrastructure
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Cryopreservation / methods*
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Cryoprotective Agents / pharmacology*
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Epigenesis, Genetic
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Fibroblasts / drug effects
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Fibroblasts / metabolism
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Fibroblasts / pathology
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Humans
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Induced Pluripotent Stem Cells / drug effects*
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Induced Pluripotent Stem Cells / metabolism
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Induced Pluripotent Stem Cells / pathology
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Motor Neurons / drug effects*
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Motor Neurons / metabolism
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Motor Neurons / pathology
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Muscular Atrophy, Spinal / genetics
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Muscular Atrophy, Spinal / metabolism
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Muscular Atrophy, Spinal / pathology
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Primary Cell Culture
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Sequence Analysis, DNA
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Transposases / chemistry
Substances
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Chromatin
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Cryoprotective Agents
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Transposases