FourCulturesCropped

Genome-wide analysis to identify pathways affecting telomere initiated senescence in budding yeast


Hsinyu Chang, Conor Lawless, Stephen G. Addinall, Morgan Taschuk, Sarah Oexle, Anil Wipat, Darren J Wilkinson and David Lydall


Supporting information data files

Manuscript (Open Access)

The full manuscript is freely available for download here.

General information

To investigate the pathways that affect entry into and exit from telomere-driven senescence, we combined a gene deletion disrupting telomerase (est1Δ) with the systematic yeast deletion collection and measured senescence in two high-throughput assays: a liquid screen and a solid screen. Our genome-wide analysis identifies genes that affect entry into and/or exit from telomere-initiated senescence and will be of interest to those studying telomere biology, replicative senescence, cancer and ageing. Our data set is complementary to other high-throughput studies relevant to telomere biology, genetic stability and DNA damage responses.

In the solid screen, 4400 yeast array deletions were crossed to an est1::NATMX strain (DLY5026) as described in Addinall et al. 2011. Double mutant cultures were passaged repeatedly on solid pins and photographs taken at every passage. Culture size at each passage was quantified using an image analysis tool Colonyzer (Lawless et al. 2010). Culture size variation with passage was used to generate quantitative senescence curves (Mean Density Profiles).

The same genetic material was used in the liquid screen, but this time passaging was carried out in liquid medium and cells were diluted and spotted onto solid agar, as described in Addinall et al. 2011, in order to assess fitness (via culture size).

Here we present various types of data captured in two senescence screens during this work.

Tips for downloading & viewing files

Note that for the .txt documents listed below in particular, it might be more useful to download these files to your hard drive before opening. The .txt documents are in tab-delimited text format, which can be viewed in a web-browser (although some users browswers have had problems rendering FileS4), however columns are more carefully aligned when opened using software with spreadsheet capabilities (such as Calc from LibreOffice, R or Microsft Excel). This will also allow you to sort the data and search through them more easily. For Windows users, to download a file, instead of viewing it in your web-browser, right-click on the link and choose "Save Link As..." (or similar). For Mac users, CTRL + click on the link and choose "Save Linked File As..." (or similar). Please note that the .tif files below really must be downloaded first, and then opened with ImageJ to view all passages. Previewing these images in a browser will only display passage 1.

Download supplementary files referenced in manuscript

Download raw image data

96 well liquid senescence assay

Four repeats of a single 384-format plate were used to assess fitness in the liquid assay. There are at least four independent biological replicates of each strain arrayed side-by-side in quadruplicate on the plate. The spatial arrangement of array deletion genotypes in this screen is presented in a visual map, which is a text-searchable .pdf file. Each tile in this document specifies the cultures row and column number (384 format) together with standard gene name and ORF y-number of their array deletion.

16 passages for 4 repeats of a 384-format plate are presented below as .tiff files, where z-stack number represents passage number. These .tiff files are best viewed with the open-source image analysis tool ImageJ. If you use the latest version, of ImageJ, a guiding grid will be visible overlaying the image. The grid can be disabled in ImageJ by Image -> Overlay -> Hide Overlay.

1536 pin solid agar senescence assay

Two spatial arrangements of the Boone SDL library were used in the solid assay, version 2 (v2) and version 3 (v3). Each version of the library contains 15 plates. Each of the two library versions has at least 4 independent biological replicates of each strain arrayed side-by side in quadruplicate.

The spatial arrangement of array deletion genotypes is presented in a visual map, which is a text-searchable .pdf file. Each tile in this document represents the location of 4 repeat cultures, specifying the quadrulicate cultures row and column number (384 format) together with the standard gene name and ORF y-number of their array deletion. This should help with locating genotypes of interest in the image stacks below.

22 passages for 15 plates in two versions of the Boone SDL library are presented below as .tiff files, where z-stack number represents passage number. These .tiff files are best viewed with the open-source image analysis tool ImageJ. If you use the latest version, of ImageJ, a guiding grid will be visible overlaying the image. The grid can be disabled in ImageJ by Image -> Overlay -> Hide Overlay.

Alternatively, the genotype present in each 1536 format row and column on each plate is identified by deletion ORF y-number in these two comma delimited text files:

Download raw data files for liquid screen

Download the quantified data for the liquid screen here:
RawDataLiquidPassage.dat (9 MB)
This tab-delimited text file is built up of 37 columns (headers included in file), named and described below. The measure of culture size used in this analysis was the "measure" column, which should be equivalent to the "trimmedgrey" column.

Download raw data files for solid screen

Download the quantified data for the solid screen here:
ChangPassageV2.dat (81 MB)
ChangPassageV3.dat (81 MB)
These tab-delimited text files are built up of 28 columns (headers not included in files), named and described below. The measure of culture size used in this analysis was the Trimmed Area column. Note that for this screen was carried out using two versions of the SDL library. The difference between the two versions is that one is spatially arrayed according to chromosome coordinate (V2) and the other is randomised (V3). These two versions were used to minimise any local competition effects on the senescence curves.