TY - JOUR
T1 - Optimized Repli-seq
T2 - improved DNA replication timing analysis by next-generation sequencing
AU - Rivera-Mulia, Juan Carlos
AU - Trevilla-Garcia, Claudia
AU - Martinez-Cifuentes, Santiago
N1 - Publisher Copyright:
© 2022, The Author(s), under exclusive licence to Springer Nature B.V.
PY - 2022/12
Y1 - 2022/12
N2 - The human genome is divided into functional units that replicate at specific times during S-phase. This temporal program is known as replication timing (RT) and is coordinated with the spatial organization of the genome and transcriptional activity. RT is also cell type–specific, dynamically regulated during development, and alterations in RT are observed in multiple diseases. Thus, the precise measure of RT is critical to understand the role of RT in gene function regulation. Distinct methods for assaying the RT program exist; however, conventional methods require thousands of cells as input, prohibiting its applicability to samples with limited cell numbers such as those from disease patients or from early developing embryos. Although single-cell RT analyses have been developed, these methods are low throughput, require generation of numerous libraries, increased sequencing costs, and produce low resolution data. Here, we developed an improved method to measure RT genome-wide that enables high-resolution analysis of low input samples. This method incorporates direct cell sorting into lysis buffer, as well as DNA fragmentation and library preparation in a single tube, resulting in higher yields, increased quality, and reproducibility with decreased costs. We also performed a systematic data processing analysis to provide standardized parameters for RT measurement. This optimized method facilitates RT analysis and will enable its application to a broad range of studies investigating the role of RT in gene expression, nuclear architecture, and disease.
AB - The human genome is divided into functional units that replicate at specific times during S-phase. This temporal program is known as replication timing (RT) and is coordinated with the spatial organization of the genome and transcriptional activity. RT is also cell type–specific, dynamically regulated during development, and alterations in RT are observed in multiple diseases. Thus, the precise measure of RT is critical to understand the role of RT in gene function regulation. Distinct methods for assaying the RT program exist; however, conventional methods require thousands of cells as input, prohibiting its applicability to samples with limited cell numbers such as those from disease patients or from early developing embryos. Although single-cell RT analyses have been developed, these methods are low throughput, require generation of numerous libraries, increased sequencing costs, and produce low resolution data. Here, we developed an improved method to measure RT genome-wide that enables high-resolution analysis of low input samples. This method incorporates direct cell sorting into lysis buffer, as well as DNA fragmentation and library preparation in a single tube, resulting in higher yields, increased quality, and reproducibility with decreased costs. We also performed a systematic data processing analysis to provide standardized parameters for RT measurement. This optimized method facilitates RT analysis and will enable its application to a broad range of studies investigating the role of RT in gene expression, nuclear architecture, and disease.
KW - DNA replication
KW - genomics
KW - library preparation
KW - next-generation sequencing
KW - replication timing
UR - http://www.scopus.com/inward/record.url?scp=85133307345&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85133307345&partnerID=8YFLogxK
U2 - 10.1007/s10577-022-09703-7
DO - 10.1007/s10577-022-09703-7
M3 - Article
C2 - 35781769
AN - SCOPUS:85133307345
SN - 0967-3849
VL - 30
SP - 401
EP - 414
JO - Chromosome Research
JF - Chromosome Research
IS - 4
ER -