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bioRxiv · 10.1101/036798

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

Pamela Milani·Renan Escalante-Chong·Brandon C. Shelley·Natasha L. Patel-Murray·Xiaofeng Xin·Miriam Adam·Berhan Mandefro·Dhruv Sareen·Clive N. Svendsen·Ernest Fraenkel
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Abstract

In recent years, the assay for transposase-accessible chromatin using sequencing (ATAC-Seq) has become a fundamental tool of epigenomic research. However, it has proven difficult to perform this technique on frozen samples because freezing cells before extracting nuclei impairs nuclear integrity and alters chromatin structure. We describe a protocol for freezing cells that is compatible with ATAC-Seq, producing results that compare well with those generated from fresh cells. We found that while flash-frozen samples are not suitable for ATAC-Seq, the assay is successful with slow-cooled cryopreserved samples. Using this method, we were able to isolate high quality, intact nuclei, and we verified that epigenetic results from fresh and cryopreserved samples agree quantitatively. We developed our protocol on a disease-relevant cell type, namely motor neurons differentiated from induced pluripotent stem cells from a patient affected by spinal muscular atrophy.

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Pamela Milani, Renan Escalante-Chong, Brandon C. Shelley, Natasha L. Patel-Murray, Xiaofeng Xin, Miriam Adam, Berhan Mandefro, Dhruv Sareen, Clive N. Svendsen, Ernest Fraenkel. 2016-01-15. Cell freezing protocol optimized for ATAC-Seq on motor neurons derived from human induced pluripotent stem cells. https://doi.org/10.1101/036798

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