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Merck

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
要旨

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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スクロース, for molecular biology, ≥99.5% (GC)
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OptiPrep 密度勾配媒体, used for cell and subcellular organelle isolation
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Triton X-100, for molecular biology
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ホスファターゼインヒビターカクテル2, aqueous solution (dark coloration may develop upon storage, which does not affect the activity)
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ヤギ血清
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塩化マグネシウム, anhydrous, ≥98%
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塩化カリウム, for molecular biology, ≥99.0%
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HEPES ナトリウム塩, ≥99.0% (titration)
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TWEEN® 20, viscous liquid
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DL-ジチオトレイトール, ≥98% (HPLC), ≥99.0% (titration)
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酢酸ナトリウム, anhydrous, for molecular biology, ≥99%
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Milli-Mark® 抗NeuN-PE抗体、クローンA60, clone A60, Milli-Mark®, from mouse
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塩化カルシウム 二水和物, BioXtra, ≥99.0%