Protocol to isolate cells in four stages of S phase for high-resolution replication-timing sequencing

Johnathan R Whetstine; Capucine Van Rechem

doi:10.1016/j.xpro.2022.101209

Protocol to isolate cells in four stages of S phase for high-resolution replication-timing sequencing

STAR Protoc. 2022 Feb 26;3(1):101209. doi: 10.1016/j.xpro.2022.101209. eCollection 2022 Mar 18.

Authors

Johnathan R Whetstine^{1

2}, Capucine Van Rechem³

Affiliations

¹ Cancer Signaling and Epigenetics Program, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
² Cancer Epigenetics Institute, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
³ Stanford Medicine Department of Pathology, Stanford, CA 94305, USA.

Abstract

Traditional replication timing (RT) experiments divide S phase into two phases: early and late. However, there is an increasing awareness that variation in RT can occur during the course of S phase and impact our understanding of RT patterns and regulation. Here, we describe a RT protocol in RPE-1 cells for collecting four phases within S and the library preparation that takes advantage of a commercial kit for methyl-DNA. This step allows BrdU-labeled DNA sequencing and assessment of RT genome wide. For complete details on the use and execution of this protocol, please refer to Van Rechem et al. (2021).

Keywords: Cell isolation; Flow Cytometry/Mass Cytometry; Genomics; Molecular Biology; Sequencing.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Bromodeoxyuridine
DNA Replication Timing*
DNA Replication* / genetics
S Phase / genetics
Sequence Analysis, DNA

Substances

Bromodeoxyuridine

Abstract

Publication types

MeSH terms

Substances

Grants and funding