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  • FACS-based isolation of fixed mouse neuronal nuclei for ATAC-seq and Hi-C.

FACS-based isolation of fixed mouse neuronal nuclei for ATAC-seq and Hi-C.

STAR protocols (2021-07-27)
Ekaterina Eremenko, Anastasia Golova, Daniel Stein, Monica Einav, Ekaterina Khrameeva, Debra Toiber
ABSTRACT

The organization of chromatin structure plays a crucial role in gene expression, DNA replication, and repair. Chromatin alterations influence gene expression, and modifications could be associated with genomic instability in the cells during aging or diseases. Here, we provide a modified protocol to isolate fixed neuronal nuclei from a single mouse cortex to investigate the spatial organization of chromatin structure on a genome-wide scale by ATAC-seq (the assay for transposase-accessible chromatin with high-throughput sequencing) and chromatin conformation by Hi-C (high-throughput chromosome conformation capture).

MATERIALS
Product Number
Brand
Product Description

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HEPES sodium salt, ≥99.0% (titration)
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DL-Dithiothreitol, ≥98% (HPLC), ≥99.0% (titration)
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Phosphatase Inhibitor Cocktail 2, aqueous solution (dark coloration may develop upon storage, which does not affect the activity)
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Potassium chloride, for molecular biology, ≥99.0%
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OptiPrep Density Gradient Medium, used for cell and subcellular organelle isolation
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Sodium acetate, anhydrous, for molecular biology, ≥99%
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TWEEN® 20, viscous liquid
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Calcium chloride dihydrate, BioXtra, ≥99.0%
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Sucrose, for molecular biology, ≥99.5% (GC)
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Goat serum
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Triton X-100, for molecular biology
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Magnesium chloride, anhydrous, ≥98%
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Milli-Mark® Anti-NeuN-PE Antibody, clone A60, clone A60, Milli-Mark®, from mouse