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Nuclei isolation of multiple brain cell types for omics interrogation.

Nature protocols (2021-01-27)
Alexi Nott, Johannes C M Schlachetzki, Bethany R Fixsen, Christopher K Glass
ABSTRACT

We present a nuclei isolation protocol for genomic and epigenomic interrogation of multiple cell type populations in the human and rodent brain. The nuclei isolation protocol allows cell type-specific profiling of neurons, microglia, oligodendrocytes, and astrocytes, being compatible with fresh and frozen samples obtained from either resected or postmortem brain tissue. This 2-day procedure consists of tissue homogenization with fixation, nuclei extraction, and antibody staining followed by fluorescence-activated nuclei sorting (FANS) and does not require specialized skillsets. Cell type-specific nuclei populations can be used for downstream omic-scale sequencing applications with an emphasis on epigenomic interrogation such as histone modifications, transcription factor binding, chromatin accessibility, and chromosome architecture. The nuclei isolation protocol enables translational examination of archived healthy and diseased brain specimens through utilization of existing medical biorepositories.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, protease free, essentially globulin free, pH 7, ≥98%
Sigma-Aldrich
Milli-Mark® Mouse IgG1-k, clone MOPC-21, Alexa Fluor 488 conjugate, Mouse IgG1-k Monoclonal Antibody control validated for use in Flow Cytometry.
Sigma-Aldrich
Anti-NeuN Antibody, clone A60, Alexa Fluor488 conjugated, clone A60, Chemicon®, from mouse
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Triton X-100, for molecular biology
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Dimethyl sulfoxide, Hybri-Max, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%