Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Summary

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

Abstract

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Introduction

For cells to successfully divide, they must first accurately and faithfully replicate their entire genome. Genome duplication occurs in a reproducible pattern, known as the DNA replication timing program¹. DNA replication timing is correlated with chromatin organization, epigenetic marks, and gene expression^2,3. Changes in replication timing occur throughout development, and are significantly related to transcriptional programs and alterations to chromatin marks and organization^4,5. Furthermore, replication timing is correlated ....

Protocol

All animals were handled in strict accordance with protocols approved by the Oklahoma Medical Research Foundation Institutional Animal Care and Use Committee.

1. Setting up adult zebrafish for breeding

1. Use a large cohort of adult male and female zebrafish of a single strain for breeding. There are small differences in the genetic makeup of zebrafish of even a single strain, use a large cohort to ensure results are representative of the genetic variability of the population and not restricted to a small subset of the population.
2. The night before embryos will be collected, place dozens of breeding adults into breeding ta....

Results

Using published replication timing data, representative replication timing profiles and quality control measures are provided¹⁵. The initial steps of processing involve aligning the sequencing data to the genome, calculating read length and genome coverage statistics, and filtering low quality, unpaired, and PCR duplicate reads. Read statistics for a typical zebrafish sequencing sample are shown in Figure 2. After filtering, read count.......

Discussion

Zebrafish provide a new and unique in vivo model system to study DNA replication timing. When timed matings are performed as detailed in this experimental protocol, thousands of embryos can be collected in a single day for experiments. These embryos develop synchronously through precisely timed and distinctly characterized stages of development. Zebrafish can be easily and accurately staged by morphology using a stereomicroscope, as zebrafish embryos develop externally and are optically clear. This protocol deta.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NaCl	Fisher Scientific	BP358-10
KCl	Fisher Scientific	P217-500
CaCl2	Fisher Scientific	C79-500
MgSO4	EMD Millipore	MMX00701
NaHCO3	Fisher Scientific	BP328-500
Pronase	Sigma	10165921001	protease solution
Phosphate buffered saline (PBS)	Sigma	D1408
Ethanol (EtOH)	KOPTEC	V1016
Bovine serum albumin (BSA)	Sigma	A9647-100G
Propidium Iodide (PI)	Invitrogen	P3566
Tris-HCl	Fisher Scientific	BP153-500
EDTA	Sigma	E9844
SDS	Santa Cruz	sc-24950
Proteinase K	NEB	P8107S
Phenol:Chloroform	Sigma	P3803-100ML
Sodium acetate	J.T.Baker	3470
Glycogen	Ambion	AM9510
RNase A	Thermo Scientific	EN0531
Quanit-iT	Invitrogen	Q33130	Reagents for fluorescence-based DNA quantification
Covaris AFA microTUBE	Covaris	520045	specialized tube for sonication
Covaris E220 Sonicator	Covaris	E220	focused ultrasonicator
Agilent 4200 Tapestation	Agilent	G2991AA	automated electrophoresis machine
D1000 ScreenTape	Agilent	5067-5582	Reagents for automated electrophoresis machine
NEBNext Ultra DNA Library Prep Kit for Illumina	NEB	Cat#E7370L	DNA library preparation kit
NEBNext Multiplex Oligos Kit for Illumina (Index Primers Set 1)	NEB	Cat#E7335S	multiplex oligos for DNA library preparation kit
NEBNext Multiplex Oligos Kit for Illumina (Index Primers Set 2)	NEB	Cat#E7500S	additional multiplex oligos for DNA library preparation kit
NEBNext Library Quant Kit for Illumina	NEB	E7630L	quantification kit for library preparation
Agencourt AMPure XP beads	Beckman Coulter	A63882	magnetic beads
Illumina HiSeq 2500	Illumina	SY–401–2501	next generation DNA sequencing platform
40 µm Falcon Nylon Cell Strainer	Fisher Scientific	08-771-1
VWR Disposable Petri Dish 100 x 25 mm	VWR	89107-632
6.0 mL Syringe for Nichiryo Model 8100	VWR	89078-446
Posi-Click Tubes, 1.7 mL, Natural Color	Denville Scientific	C2170 (1001002)	Dnase/Rnase free
Vortex Genie 2	Scientific Industries	SI-0236
Wash Bottles	VWR	16650-022	Low-Density Polyethylene, Wide Mouth
Strainer	VWR	470092-440	6.9 cm, fine mesh
Corssing tank	Aquaneering	ZHCT100	individual breeding tank
iSpawn	Techniplast	N/A	large breeding tank
FACSAria II	BD biosciences	N/A	cell sorting machine
Wild M5a steromicroscope	Wild Heerbrugg	N/A	dissecting microscope
Qubit 3 Fluorometer	Thermo Scientific	Q33216	quantitative fluorescence-based method for determining DNA concentration
Matlab	Mathworks	version 2017a
Matlab Statistics Toolbox	Mathworks	version 11.1
Matlab Curve Fitting Toolbox	Mathworks	version 3.5.5