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

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).

Graphical abstract

Figure 1

Quality Control for Efficient Fragmentation of Adaptor-Ligated Genomic DNA Gel view of denatured, adaptor-ligated genomic yeast DNA before fragmentation and after 2 or 4 cycles of sonication, analyzed by Agilent RNA Screen Tape. The sample sonicated for 4 cycles shows an appropriate size distribution. The low molecular weight marker band is situated at 25 nt.

Figure 2

Quality Control for Efficient Capture of Fragmented DNA Gel view of genomic yeast DNA fragments before (Input) and after (Bound) capture on Streptavidin beads, analyzed by Agilent RNA Screen Tape. The low molecular weight marker band is situated at 25 nt.

Figure 3

Quality Control for Efficient Conversion of Captured ssDNA to dsDNA Gel view of captured DNA fragments after second strand synthesis, analyzed on an Agilent High Sensitivity D1000 ScreenTape. The lane labeled “Control” shows the faint signal arising from a sample of undigested yeast genomic DNA and the lane labeled “Treatment” shows the abundant material derived from a sample of yeast genomic DNA digested with a restriction enzyme. The low and high molecular weight marker bands are situated at 25 and 1500 bp, respectively. Note that the original ssDNA present in the sample is not well detected on this type of dsDNA-selective ScreenTape.

Figure 4

Quality Control for Efficient Ligation of the Distal Adaptor and Correct Size Range Gel view of DNA libraries after ligation of the distal adaptor, analyzed on an Agilent High Sensitivity D1000 ScreenTape. As in Figure 3, the two lanes show material generated from a sample of undigested DNA (Control) and from a sample digested with a restriction enzyme (Treatment). The low and high molecular weight marker bands are situated at 25 and 1500 bp, respectively.

Figure 5

Quality Control for Efficient Amplification of the Library The electropherogram shows GLOE-Seq libraries of high (top) and low (bottom) quality, analyzed by Agilent Bioanalyzer High Sensitivity DNA Kit. (AD: adaptor dimers; LM: lower marker; UM: upper marker). Note that the size range of the libraries shown here slightly exceeds the recommended value; however, a deviation of this magnitude is uncritical.

Figure 6

Flowchart of GLOE-Pipe