Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Summary

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

Abstract

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.

This article describes in detail the "Copy Number Ratio of S/G1 for mapping genomic Time of Replication" method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

Introduction

Mammalian DNA replication is tightly regulated to ensure the precise replication of each chromosome exactly once during the cell cycle. Replication occurs according to a highly regulated order — multiple large genomic regions (~Mb) replicate at the beginning of S phase (early replicating domains) whereas other genomic regions replicate later at middle or late S phase (middle and late replicating domains)¹. Most of the genome replicates at the same time in all tissues (constitutive ToR domains), whereas 30% - 50% of the genome, changes its ToR between tissues², during differentiation^3,

Protocol

Note: ToR can be measured only on growing, unsynchronized cells. The procedure should begin with at least 1 - 2 x 10⁶ fast growing cells, which will usually result in ~1 x 10⁵ cells in S phase (the rate limiting step). It is recommended to conduct each experiment using two or three replicates. The entire process of CNR-ToR can be completed within one week — two days should be dedicated to all steps up to library preparation, one to two days are needed for sequencing and an additional day is necessary for the initial data analysis.

1. Collection of Cells from Culture

NOTE: The protocol is writt....

Results

A typical ToR map is shown in Figure 3 for mouse embryonic fibroblasts (MEFs). This figure demonstrates the analysis process since it shows both the dots, which are the normalized S/G1 ratio for individual windows (step 8.3), as well as the line which results from the cubic smoothing and interpolation (step 8.5).

Such maps capture the organization of the replication program, which is a patchwork of two types of .......

Discussion

CNR-ToR can be performed in principle on any eukaryotic proliferating cell population that can be divided by FACS to S and G1 phases (reviewed by Rhind N. and Gilbert DM²⁰). The method described here has been adjusted to mammalian cells with a genome size of ~3 Gb such as human and mouse. Small changes in the CNR-ToR protocol (in cell preparation and sequencing depth) are needed, in order to adjust it to other eukaryotes. Attention must be paid to the collection of sufficient amounts of S phase ce.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PBS	BI (Biological Industries)	02-023-1A
Trypsin-EDTA	BI (Biological Industries)	03-052-1B
15 mL conical tube	Corning	430790
5 mL Polystyrene round Bottom tube with cell strainer cap	BD-Falcon	352235
Ethanol	Gadot	64-17-5
RNAse-A 10 mg/mL	Sigma	R4875
Propidiom iodide 1 mg/mL	Sigma	P4170
Parafilm	Parafilm	PM-996
1.5 mL DNA LoBind Eppendorf tubes	Eppendorf	22431021
BSA	Sigma	A7906
1.7 mL MaxyClear tube	Axygen	MCT-175-C
magnetic beads - Agencourt AMPure XP	Beckman Coulter	A63881
Ultrasonicator	Covaris	M-series  -530092
50 µL microTUBE AFA Fiber Screw-Cap 6 x 16 mm	Covaris	520096
Qubit fluorometer	Invitrogen
Qubit dsDNA High Sensitivity (HS) Assay Kit	Invitrogen	Q32854
Electrophoresis 2200 Tape station system	Agilent	D1000 ScreenTape
Seqeuncing - Illumina NextSeq system	Illumina	SY-415-1001
Dneasy kit for DNA purification	Qiagen	69504
PureProteom Magnetic Stand	Millipore	LSKMAGS08
Anti-BrdU/FITC	DAKO	F7210
FACS sorter	BD	FACSARIA III
FACS software	BD	FACSDiva v 8.0.1