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URL: https://dx.doi.org/DOI:10.17504/protocols.io.qmmdu46

Authors: Nicholas Leigh

Summary: This protocol is designed to obtain blastema cells (and wound epidermis if left attached) into a single cell suspension. It was specifically designed for single cell RNA sequencing, but can be used for other applications in which a single cell suspension is desired. The goal is to obtain a single cell suspension with near 100% viability in a rapid fashion. This protocol does not use centrifugation, but instead uses a gradient replacement strategy pioneered by Briggs et al, 2018 Science. This strategy washes away free-floating RNA from cells by using progressively denser solutions to "lift" off the solution in which the cells are currently emersed. This protocol could be can be modified to utilize centrifugation instead and likely should be if planning on anything other than single cell RNAseq on inDrops as a next step.

Associated Publications: Leigh ND, Dunlap GS, Johnson K, Mariano R, Oshiro R, Wong AY, Bryant DM, Miller BM, Ratner A, Chen A, Ye WW, Haas BJ, Whited JL, Transcriptomic landscape of the blastema niche in regenerating adult axolotl limbs at single-cell resolution. Nature Communications doi: 10.1038/s41467-018-07604-0

Affiliations: Brigham and Women's Hospital/Harvard Medical School

External URL: https://doi.org/10.1038/s41467-018-07604-0

Version: 1

Publication Date: 2018

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