Quantitative FISH (qFISH)

For telomere length determination, fluorescence in situ hybridization (FISH) was utilized in a quantitative manner⁸⁸. The staining protocol was optimized after the work of Seo and Lee⁸⁹. Per strain, 100 gravid adults were picked to an unseeded small NGM plate to remove the majority of OP50 bacteria. From there, worms were picked to a 5 µl drop of Egg buffer (25 mM HEPES/KOH pH 7.4, 118 mM NaCl, 48 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 1% Tween-20) on a cover slip and dissected using 20 gauge needles (Sterican, Roth #C718.1) to release embryos and gonads. The samples were fixed by adding 5 µl of 2% Formaldehyde solution and incubating for 5 min. To remove the Formaldehyde solution, samples were washed on the cover slip by adding and removing Egg buffer carefully by pipetting. For permeabilization of the cuticle, the worms were afterwards treated by freeze cracking⁹⁰. The cover slips were put on a Poly-lysine coated slide (Sigma Aldrich, #P0425) and the slides transferred to an aluminum block on dry ice for freezing. After 15 min freezing on the aluminum block, the cover slips were removed and the slides immersed first in ice-cold methanol, then in ice-cold acetone for 5 min, respectively. To remove the solutions the slides were washed in 1x PBS (10 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl) for 15 min. For additional permeabilization the samples were incubated in permeabilization buffer (20 mM Tris/HCl pH 7.5, 50 mM NaCl, 3 mM MgCl₂, 300 mM Sucrose, 0.5% Triton X-100) at 37 °C for 30 min followed by a wash in 1x PBS for 5 min at room temperature. To prevent unspecific binding of the FISH probe, the samples were treated with 20 µl RNase A solution (1x PBS, 0.1% Tween-20, 10 µg/ml RNase A) at 37 °C for 1 h in a humid chamber. Afterwards the slides were washed in 1x PBS-T (1x PBS, 0.1% Tween-20) for 10 min at room temperature and dehydrated by successive 3 min washes in 70%, 85 and 100% ethanol and air dried. For pre-hybridization 50 µl of hybridization solution (3X SSC, 50% Formamide, 10% (w/v) Dextran-Sulfate, 50 µg/ml Heparin, 100 µg/ml yeast tRNA, 100 µg/ml sheared salmon sperm DNA) were added to the sample and the slides incubated in a humid chamber for 1 h at 37 °C. The FISH probe (PNA-FISH TTAGGC telomeric probe, Panagene, resuspended to 100 µM, fluorophore: Alexa-555) was prepared as a 1:500 dilution in hybridization solution and denatured for 5 min at 70 °C. After pre-hybridization, the solution on the slides was removed as much as possible by pipetting and 20 µl of FISH probe were added, then covered by a cover slip. For hybridization of the probe the slides were denatured on a heat block prepared with wet paper towels for humidity at 80 °C for 3 min and transferred to a humid chamber for incubation overnight at 37 °C. The next day the slides were washed twice in 1x PBS-T for 5 min to remove the probe. To fixate the staining, the samples were incubated in hybridization wash solution (2X SSC, 50% Formamide) for 30 min at 37 °C. As a last step the slides were washed in 1x PBS-T twice for 15 min at room temperature and mounted by adding 10–20 µl Vectashield mounting medium containing DAPI (Vector laboratories, #H-1200-10). The pictures were taken with a Leica TCS SP5 confocal microscope (objective: CX PL APO CS 63sx oil NA: 1.4, pinhole 60.05 µm, 2x zoom, PMT detectors, acquisition software Leica LAS AF). The images stacks were composed by a sequence of pictures acquired every 0.5 µm on the z-axis. The laser and gain settings were adjusted according to the sample with the lowest FISH intensity. For analysis, images were opened in Image J/Fiji and the channels split into the DAPI and red channel. A mask of the image was created to infer the volume of the imaged object. The threshold function of the software was used with activated plugins for identification of round objects (Otsu). After setting the threshold for the image in the histogram settings, the z-stack was converted to a binary mask and using the 3D OC Options menu volume, mean gray values and integrated density of the FISH foci were calculated. Additionally, the 3D Object counter menu was used and the filters set to a minimum of 2. The values obtained by this analysis were averaged over several images of either germlines or embryos of the same strain and used for quantitative comparison of telomere length. For comparison, all values obtained for the mutant strains were scaled relative to the average of the wild type values. The barplots were created using R with standard and publicly available scripts (RColorBrewer-v 1.1-2, ggpubr-v 0.4.0, plyr-v 1.8.6, viridis-v 0.5.1, viridisLite-v 0.3.0, ggforce-v 0.3.2, ggsignif-v 0.6.0, dplyr-v 1.0.2, ggplot2-v 3.3.3, readr-v 1.4.0).