Cell freezing protocol suitable for ATAC-Seq on motor neurons derived from human induced pluripotent stem cells

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 alte...

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Bibliographic Details
Published in:Scientific reports 2016-05, Vol.6 (1), p.25474-25474, Article 25474
Main Authors: Milani, Pamela, Escalante-Chong, Renan, Shelley, Brandon C., Patel-Murray, Natasha L., Xin, Xiaofeng, Adam, Miriam, Mandefro, Berhan, Sareen, Dhruv, Svendsen, Clive N., Fraenkel, Ernest
Format: Article
Language:English
Subjects:
631/1647/514/2254
631/61/212/177
Chromatin
Cryopreservation
Freezing
Humanities and Social Sciences
Motor neurons
Motor task performance
multidisciplinary
Neuromuscular diseases
Pluripotency
Science
Science (multidisciplinary)
Spinal muscular atrophy
Stem cells
Transposase
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Summary: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.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep25474