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

Autor:	Milani P; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139, United States of America., Escalante-Chong R; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139, United States of America., Shelley BC; Board of Governors-Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, 90048, United States of America., Patel-Murray NL; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139, United States of America., Xin X; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139, United States of America., Adam M; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139, United States of America., Mandefro B; Board of Governors-Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, 90048, United States of America.; iPSC Core - The David and Janet Polak Foundation Stem Cell Core Laboratory, California, 90048, United States of America., Sareen D; Board of Governors-Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, 90048, United States of America.; iPSC Core - The David and Janet Polak Foundation Stem Cell Core Laboratory, California, 90048, United States of America.; Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, 90048, United States of America., Svendsen CN; Board of Governors-Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, 90048, United States of America.; iPSC Core - The David and Janet Polak Foundation Stem Cell Core Laboratory, California, 90048, United States of America.; Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, 90048, United States of America., Fraenkel E; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139, United States of America.
Jazyk:	angličtina
Zdroj:	Scientific reports [Sci Rep] 2016 May 05; Vol. 6, pp. 25474. Date of Electronic Publication: 2016 May 05.
DOI:	10.1038/srep25474
Abstrakt:	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.
Databáze:	MEDLINE
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