Year: 2010
Type: article
Abstract: Analyses of replication timing in various cell types have yielded insights into genome organization and repackaging events during development, suggesting an important role for the timing program itself or 3D genome organization in regulating developmental gene expression [1][2][3] .Although the mechanisms that specify the timing and placement of origin ring in higher eukaryotes remain a mystery, all eukaryotes have a de ned replication timing program that is largely conserved between closely related species 4 , including human and mouse 3,5 .Here, we describe details of the methodology used to analyze replication timing genome-wide in mammalian cells.In this protocol, asynchronously cycling cells are pulse labeled with the nucleotide analog 5-bromo-2-deoxyuridine \(BrdU) and then sorted into S-phase fractions based on DNA content using ow cytometry.BrdU-labeled DNA from each fraction is immunoprecipitated, ampli ed, differentially labeled, and co-hybridized to a whole-genome CGH microarray.To address the analysis bottleneck that often follows genomic experiments, the second half of the protocol covers methods to analyze array data produced using the R framework for statistical computing [6][7][8] , including normalization, scaling, and data quality measures, loess \(local polynomial) smoothing of replication timing values, segmentation of data into domains, and assignment of timing values to gene promoters.After these preprocessing steps, we cover various common downstream analyses, such as k-means and hierarchical clustering, and methods to relate changes in the replication program to gene expression and other genetic and epigenetic datasets.Additional details and descriptions of the method can be found at www.ReplicationDomain.org/Protocol.Reagents • 1mg/mL BrdU \(5-bromo-2'-deoxyuridine)\(Sigma Aldrich, B5002) \(All solutions are in ddH 2 0 unless otherwise indicated) • Cell culture medium appropriate for cell type • 1 X PBS \(10 X Phosphate Buffered Saline, 137 mM NaCl , 2.7mMKCl,10mM Na 2 HPO 4 , 2mM KH 2 PO 4 , diluted with dH 2 O) • 0.2X Trypsin-EDTA \(diluted with PBS from a 1X solution, Mediatech 25-053-Cl) or Accutase \(Innovative Cell Technologies AT104) • Fetal Bovine Serum \(FBS) • DAPI • 1 mg/mL Propidium Iodide\(PI, Sigma P4179-100MG dissolved in ddH 2 O and ltered) • 10 mg/mL RNase A • SDS-PK buffer \(dH 2 O with 50mM Tris-HCl, pH8.0/ 10mM EDTA/ 1M NaCl/ 0.5%SDS) • 20mg/mL Proteinase K \(dissolved in ddH 2 O) • 20mg/mL Glycogen \(Fermentas RO561) • Tris-saturated Phenol-chloroform • Chloroform
Source: Protocol Exchange
Institutions: Florida State University, National Institute of Genetics
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
OpenAlex ID: W2093489526