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Dr. Ebtesam Hassan Omar Nafe :: Publications:

Genome-wide analysis of replication timing by next-generation sequencing with E/L Repli-seq
Authors: Claire Marchal1,4, Takayo Sasaki1,4, Daniel Vera2 , Korey Wilson1, Jiao Sima1, Juan Carlos Rivera-Mulia1,Claudia Trevilla-García1, Coralin Nogues1, Ebtesam Nafie3 & David M Gilbert1,2
Year: 2018
Keywords: DNA replicationGenome informaticsGenomic analysisNext-generation sequencing
Journal: Nature Protocols
Volume: 13
Issue: 2018
Pages: 819–839
Publisher: Springer Nature
Local/International: International
Paper Link: Not Available
Full paper Ebtesam Hassan Omar Nafe_nprot.2017.148.pdf
Supplementary materials Not Available

Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early- and late-replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and subnuclear position. Moreover, RT is regulated during development and is altered in diseases. Here, we describe E/L Repli-seq, an extension of our Repli-chip protocol. E/L Repli-seq is a rapid, robust and relatively inexpensive protocol for analyzing RT by next-generation sequencing (NGS), allowing genome-wide assessment of how cellular processes are linked to RT. Briefly, cells are pulse-labeled with BrdU, and early and late S-phase fractions are sorted by flow cytometry. Labeled nascent DNA is immunoprecipitated from both fractions and sequenced. Data processing leads to a single bedGraph file containing the ratio of nascent DNA from early versus late S-phase fractions. The results are comparable to those of Repli-chip, with the additional benefits of genome-wide sequence information and an increased dynamic range. We also provide computational pipelines for downstream analyses, for parsing phased genomes using single-nucleotide polymorphisms (SNPs) to analyze RT allelic asynchrony, and for direct comparison to Repli-chip data. This protocol can be performed in up to 3 d before sequencing, and requires basic cellular and molecular biology skills, as well as a basic understanding of Unix and R.

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