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

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.

SUBMITTER: Milani P 

PROVIDER: S-EPMC4857123 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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

Milani Pamela P   Escalante-Chong Renan R   Shelley Brandon C BC   Patel-Murray Natasha L NL   Xin Xiaofeng X   Adam Miriam M   Mandefro Berhan B   Sareen Dhruv D   Svendsen Clive N CN   Fraenkel Ernest E  

Scientific reports 20160505

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 cel  ...[more]

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