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  • Preparation and Analysis of GLOE-Seq Libraries for Genome-Wide Mapping of DNA Replication Patterns, Single-Strand Breaks, and Lesions.

Preparation and Analysis of GLOE-Seq Libraries for Genome-Wide Mapping of DNA Replication Patterns, Single-Strand Breaks, and Lesions.

STAR protocols (2020-10-29)
Giuseppe Petrosino, Nicola Zilio, Annie M Sriramachandran, Helle D Ulrich
ABSTRACT

GLOE-Seq is a next-generation sequencing method for the genome-wide mapping of 3'-OH termini, either resulting from single- or double-strand breaks or introduced by enzymatic conversion of lesions or modified nucleotides. This protocol provides instructions for isolation of genomic DNA from budding yeast or mammalian cells, preparation of libraries for sequencing, and data analysis by the associated computational pipeline, GLOE-Pipe. It is optimized for the Illumina next-generation sequencing platform and can be adapted to intact genomic DNA of any origin. For complete details on the use and execution of this protocol, please refer to Sriramachandran et al. (2020).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium hydroxide, BioXtra, ≥98% (acidimetric), pellets (anhydrous)
Sigma-Aldrich
Sodium chloride, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
Sigma-Aldrich
Sodium citrate dihydrate, ≥99%, FG
Sigma-Aldrich
2-Mercaptoethanol, for molecular biology, suitable for electrophoresis, suitable for cell culture, BioReagent, 99% (GC/titration)
Sigma-Aldrich
D-Sorbitol, ≥98% (GC)
Sigma-Aldrich
Poly(ethylene glycol), BioUltra, 8,000
Sigma-Aldrich
N-Lauroylsarcosine sodium salt, BioUltra, for molecular biology, ≥99.0% (HPLC)
Sigma-Aldrich
Ethylenediaminetetraacetic acid, anhydrous, crystalline, BioReagent, suitable for cell culture
Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥98.5% (GC)