Genome-wide mapping of DNA double-strand breaks from eukaryotic cell cultures using Break-seq

Abstract

We describe a genome-wide DNA double-strand break (DSB) mapping technique, Break-seq. In this protocol, we provide step-by-step instructions for cell embedment in agarose, in-gel DSB labeling and subsequent capture, followed by standard Illumina library construction and sequencing. We also provide the framework for sequence data processing and DSB peak identification. Finally, we present a custom-designed 3D-printed device for processing agarose-embedded DNA samples. The protocol is applicable to Saccharomyces cerevisiae, as well as mammalian suspension, adherent, and 3D organoid cell cultures. For complete details on the use and execution of this protocol, please refer to Hoffman et al. (2015) and Chakraborty et al. (2020).

Keywords: Genomics; Model Organisms; Molecular Biology; Sequencing.

Graphical abstract

Figure 1

Preparation of frozen EDTA solution for harvesting yeast culture An autoclaved 500-mL Nalgene bottle (A) is used to prepare 40 mL frozen 0.2 M EDTA by storage in a -20°C freezer (B–D).

Figure 2

Comparison of agarose plugs prepared from yeast versus human cells Demonstration of air bubbles trapped in agarose causing structural damage to the agarose plugs during and after plug casting, respectively.

Figure 3

Humidity chamber(s) used for processing agarose plugs during labeling reactions and subsequent washing (A–F) The figure shows a 3D printed design (A–E) and a jerry-rigged design if a 3D printer is not available (F). The components of the 3D printed humidity chamber include: a base which holds the buffer (A), a porous tray with a handle where the agarose plug (3D printed to life size 0.5 cm × 1 cm × 0.1 cm in green color) is placed, which fits into the base (B), a cap for the base and tray for maintaining humidity (C). (D) A multi-chamber design which contains three bases fitted with three individual trays and caps and connected to a pump. (E) A peristaltic pump which circulates solution through the multi-chambers. The directions of influx and efflux, are marked as “in” and “out”, respectively. (F) A jerry-rigged humidity chamber using a 1-mL tip box as the base and the insert from a 1-mL tip box as the tray. (G) A real agarose plug placed in the center of the parafilm laid on the platform of the jerry-rigged humidity chamber, without (left) and with (right) 100 μL End-repair reaction buffer. Note that the buffer surrounds the agarose plug and stays “hovered” with surface tension.

Figure 4

Exemplary agarose gel images of DNA resulted from step 4.h, step 9.d, and step 10.p for the same sample and examples for problems described in Problem 2 (A) Fragmented DNA to ~400 bp average size after sonication. The 100-bp DNA ladder is shown in (A), and only marked for other panels for clarity. (B) PCR amplified DNA after adapter ligation (note the shift of average size of DNA to 500–600 bp) with noticeable level of free adapters after PCR. (C) AMPure purified DNA library (lane 1) and flow-through containing free adapters (lane 2). Examples for problems described in Problem 2 are also presented (D-F). (D) Unfragmented DNA after sonication. Lane 1, a sample with partially fragmented DNA; lane 2, a sample with no fragmentation at all. (E) Low level of PCR product. Lane 1, a sample with low level of amplification of DNA. Lane 2, a sample with normal level of amplification of DNA as comparison. (F) Retention of free adapters in the DNA library after AMPure purification . Lane 1, a sample after AMPure purification. Lane 2, flow-through of the sample in lane 1 after AMPure purification.