Yeast Genomic DNA Extraction and Replication Timing Analysis

Yeast genomic DNA was extracted using the spheroplast method (http://fangman-brewergeneticswashingtonedu/indexhtml). Samples were prepared according to the TruSeq Nano sample preparation guide from Illumina. To generate replication timing profiles, the ratio of uniquely mapped reads in the replicating samples to the nonreplicating samples was calculated following Müller et al. (2014) (link), and profiles were smoothed by a Fourier transformation (Müller et al. 2014 (link)). A replication peak was defined as a curve point where the S to G1 ratio/Δkb changed from plus to minus and the same sign was kept at more than 3 kb from the change point. A peak is therefore defined as a local maximum. The values of T_rep were from OriDB (Siow et al. 2012 (link)).

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Morafraile E.C., Hänni C., Allen G., Zeisner T., Clarke C., Johnson M.C., Santos M.M., Carroll L., Minchell N.E., Baxter J., Banks P., Lydall D, & Zegerman P. (2019). Checkpoint inhibition of origin firing prevents DNA topological stress. Genes & Development, 33(21-22), 1539-1554.

Publication 2019

TruSeq Nano

Genomic Replication Spheroplast Yeast

Corresponding Organization :

Other organizations : Wellcome/Cancer Research UK Gurdon Institute, Wellcome Trust, University of Sussex, Newcastle University

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Variable analysis

independent variables

Yeast genomic DNA extraction method (spheroplast method)

dependent variables

Replication timing profiles calculated as the ratio of uniquely mapped reads in the replicating samples to the nonreplicating samples
Identification of replication peaks defined as a curve point where the S to G1 ratio/Δkb changed from plus to minus and the same sign was kept at more than 3 kb from the change point
Replication timing (Trep) values from OriDB

control variables

Sample preparation according to the TruSeq Nano sample preparation guide from Illumina
Smoothing of replication timing profiles by Fourier transformation (as described in Müller et al. 2014)

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