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

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

Nature protocols (2020-11-25)
Hisashi Miura, Saori Takahashi, Takahiro Shibata, Koji Nagao, Chikashi Obuse, Katsuzumi Okumura, Masato Ogata, Ichiro Hiratani, Shin-Ichiro Takebayashi
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ribonuclease A from bovine pancreas, for molecular biology, ≥70 Kunitz units/mg protein, lyophilized
Sigma-Aldrich
SeqPlex DNA Amplification Kit, For use with high throughput sequencing technologies, Whole Genome Amplification kit designed to facilitate Next Gen Sequencing.
Sigma-Aldrich
Proteinase K from Tritirachium album, buffered aqueous glycerol solution, for molecular biology, ≥800 units/mL