論文

国際誌
2020年12月

Mapping replication timing domains genome wide in single mammalian cells with single-cell DNA replication sequencing.

Nature protocols
  • Hisashi Miura
  • ,
  • Saori Takahashi
  • ,
  • Takahiro Shibata
  • ,
  • Koji Nagao
  • ,
  • Chikashi Obuse
  • ,
  • Katsuzumi Okumura
  • ,
  • Masato Ogata
  • ,
  • Ichiro Hiratani
  • ,
  • Shin-Ichiro Takebayashi

15
12
開始ページ
4058
終了ページ
4100
記述言語
英語
掲載種別
研究論文(学術雑誌)
DOI
10.1038/s41596-020-0378-5

Replication timing (RT) domains are stable units of chromosome structure that are regulated in the context of development and disease. Conventional genome-wide RT mapping methods require many S-phase cells for either the effective enrichment of replicating DNA through bromodeoxyuridine (BrdU) immunoprecipitation or the determination of copy-number differences during S-phase, which precludes their application to non-abundant cell types and single cells. Here, we provide a simple, cost-effective, and robust protocol for single-cell DNA replication sequencing (scRepli-seq). The scRepli-seq methodology relies on whole-genome amplification (WGA) of genomic DNA (gDNA) from single S-phase cells and next-generation sequencing (NGS)-based determination of copy-number differences that arise between replicated and unreplicated DNA. Haplotype-resolved scRepli-seq, which distinguishes pairs of homologous chromosomes within a single cell, is feasible by using single-nucleotide polymorphism (SNP)/indel information. We also provide computational pipelines for quality control, normalization, and binarization of the scRepli-seq data. The experimental portion of this protocol (before sequencing) takes 3 d.

リンク情報
DOI
https://doi.org/10.1038/s41596-020-0378-5
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/33230331
ID情報
  • DOI : 10.1038/s41596-020-0378-5
  • PubMed ID : 33230331

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