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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
RESUMEN

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).

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Sacarosa, for molecular biology, ≥99.5% (GC)
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Medio para gradiente de densidad OptiPrep, used for cell and subcellular organelle isolation
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Triton X-100, for molecular biology
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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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Suero de cabra
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Magnesium chloride, anhydrous, ≥98%
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Cloruro de potasio, for molecular biology, ≥99.0%
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TWEEN® 20, viscous liquid
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HEPES sodium salt, ≥99.0% (titration)
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DL-Ditiotreitol, ≥98% (HPLC), ≥99.0% (titration)
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Sodium acetate, anhydrous, for molecular biology, ≥99%
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Anticuerpo Milli-Mark® Anti-NeuN-PE, clon A60, clone A60, Milli-Mark®, from mouse
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Cloruro de calcio dihydrate, BioXtra, ≥99.0%