Jan 08, 2020

Public workspaceNuclei Isolation for SNARE-seq2

DOI
dx.doi.org/10.17504/protocols.io.8tvhwn6
  • 1University of California, San Diego, and Sanford Burnham Prebys Medical Discovery Institute;
  • 2Sanford Burnham Prebys Medical Discovery Institute
  • BICCN / BICAN
Carter Palmer
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DOI: dx.doi.org/10.17504/protocols.io.8tvhwn6
Protocol Citation: Carter Palmer, Christine Liu, Jerold Chun 2020. Nuclei Isolation for SNARE-seq2 . protocols.io https://dx.doi.org/10.17504/protocols.io.8tvhwn6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 29, 2019
Last Modified: January 08, 2020
Protocol Integer ID: 29269
Keywords: Brain, Nuclei, Isolation, Single-Cell
Abstract
This protocol is intended to be used for the isolation of nuclei from fresh-frozen brain tissue in preparation for analysis by Single-Nucleus Chromatin Accessibility and mRNA Expression sequencing (SNARE-seq). It has been applied to tissues from mouse, marmoset, and human.
Guidelines
This protocol is designed specifically for isolating tissue for SNARE-seq. RNA stability is considered at every step. Samples are kept on ice throughout the process, all centrifuges are pre-chilled to 4 oC before use, and RNase Away is used to spray down all surfaces and pipets before use.
Materials
MATERIALS
ReagentSucroseFisher ScientificCatalog #S3-212
ReagentProtease Inhibitor Tablets cOmplete Mini EDTA free RocheCatalog #11836170001
ReagentRNase InhibitorTakara Bio Inc.Catalog #2313A
ReagentBovine Serum AlbuminGemini Bio-ProductsCatalog #700-107P
ReagentMagnesium acetate tetrahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M5661
ReagentCalcium chloride dihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #C5080
ReagentEthylenediaminetetraacetic acid (EDTA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #EDS
ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787
Reagent16% Formaldehyde (w/v) Methanol-freeThermo Fisher ScientificCatalog #28906
ReagentDAPIMerck MilliporeSigma (Sigma-Aldrich)Catalog #10236276001
Reagent50um filtersSysmexCatalog #04-0042-2317
ReagentTissue HomogenizerCatalog #358005