(a) DIC photomicrograph of eggs obtained from FUDR (120 µM final) treated animals. Eggs are terminally arrested around the ventral closure (“bean stage”, 400–500 nuclei) and show a shrunken cell mass. Birefringent gut granules are observed in the middle of the eggs. Images were taken ~48 hr after FUDR treatment. (Scale bar = 20 µm). (b) Number of adult worms that produce eggs 24 hr after FUDR treatment. Of the 298 worms evaluated, all of the animals developed germline with eggs inside. (c) Treatment with FUDR dramatically reduces the RNA content in eggs. Total RNA was extracted from FUDR-treated whole wt (N2) worms, from eggs isolated from FUDR-treated N2 worms after ~28 hr of FUDR treatment and from eggs from non-FUDR-treated N2 worms (the same time point as the RNA-seq young reference),. ***P<0.001, comparison between whole worms and eggs treated with FUDR, unpaired t-test, n=3, Error bars S.E.M; ##P<0.01, comparison between eggs treated with FUDR and no FUDR, unpaired t-test, n=3, Error bars S.E.M. (d) Electrophoresis of RNA extracted from whole worms or eggs isolated from FUDR treated animals. Same number of animals used for each sample. Comparison of equal volumes (10 µl) of total RNA loaded from FUDR-treated whole worms and eggs isolated from FUDR-treated animals, resolved in an agarose gel. (e) Original drift plot from Figure 2a is shown again for comparison. Note that box in the middle of the drift plot, which is a Tukey-plot, represents the interquartile mean, or 50% of the transcriptome that changes less with age. As drift is also observed in the interquartile mean, drift is not driven by extreme outliers, but by the majority of the genes across the entire transcriptome. (f) Drift plot generated from our data-set only including genes that were also detected in the CF512 sterile strain data-set from (Murphy et al., 2003). (g) Drift plot generated after removing 7,292 genes involved in egg-related functions detected from an eggs-only RNA-seq data-set (Osborne Nishimura et al., 2015). (h) DIC photomicrograph of eggs obtained from untreated and FUDR-treated animals carrying the Pgcy-8::GFP reporter for AFD neurons. (i) Fluorescence microscopy images show AFD neurons in eggs derived from untreated adults (left panel, white arrows) but not in eggs obtained from FUDR-treated adults (middle panel), confirming that FUDR treated eggs do not progress past the “bean stage”. FUDR does not inhibit Pgcy-8::GFP expression in adults (right panel). (j) Overlay of h and i. (k) Drift plots using our data-set including only the genes that are highly enriched in AFD, ASE or NSM neurons (Etchberger et al., 2007; Spencer et al., 2014). As FUDR arrests embryonic development before the birth of these neurons, the drift-plots cannot be influenced by RNA derived from eggs. Explanations for Figure 2—figure supplement 2 In the experiments presented in the main manuscript, we used FUDR to sterilize the animals from which we subsequently extracted RNA for RNA-seq. Thus, our samples contained fractions of egg RNA. The following control experiments and analysis show that the fraction of RNA in our samples coming from eggs is small and does not influence the phenomenon of transcriptional drift and its attenuation by mianserin. We first isolated eggs from FUDR-treated and untreated animals. FUDR treatment causes the cell mass inside the eggs to shrink and to terminally arrest at around bean stage (400–500 nuclei) (Figure 2—figure supplement 2a). FUDR-treated animals all contained similar numbers of eggs 24 hr after FUDR treatement (n=298) (Figure 2—figure supplement 2b) Note that many of the reported FUDR side-effects such as a lack of germline are not observed in 96-well liquid culture (Gomez-Amaro et al., 2015). Extracting RNA from whole worms or eggs isolated from whole worms showed that FUDR-treated eggs contained 5 times less RNA compared to untreated eggs. The fraction of RNA originating from the eggs in FUDR-treated worms was roughly ~5% (Figure 2—figure supplement 2c,d). We next asked whether this fraction could in anyway influence the phenomenon of transcriptional drift. The original plots (Figure 2a, or Figure 2—figure supplement 2e) of the entire transcriptome show that drift-variance increases in the interquartile mean (boxes) showing that it is not driven by a set of outlier genes, making it unlikely that the 5% fraction would influence drift-variance (Krzywinski and Altman, 2014). Nevertheless, to test possible interference, we calculated drift plots for various subsets of our data excluding transcrips expressed in eggs. The Murphy data were derived from CF512 (sterile) animals and thus any genes detected do not originate from eggs. We therefore excluded all genes not detected by Murphy et al from our data-set and recalculated drift. The resulting drift plot still shows a dramatic increase in drift-variance and attenuation by mianserin (Figure 2—figure supplement 2f). A potential problem with the approach used in Figure 2—figure supplement 2f is that it only removed eggs/germline genes that are specific for eggs but that it did not remove genes that are present in both eggs and soma. We therefore removed all genes that were identified in C. elegans eggs by RNA-seq from our data-set to plot Figure 2—figure supplement 2g (Osborne Nishimura et al., 2015). Of the 7,700 transcripts identified in eggs, 7,200 were present in our data-set. Note that this approach removes all ubiquitously expressed genes like ribosomal, mitochondrial and similar housekeeping genes that are present in both embryos and soma. Even though this operation removes only 7,200 out of 19,196 individual genes present in the data-set, these 7,200 genes account for 73% of total mRNA counts. Despite this dramatic reduction in overall mRNA transcripts, the drift plot combining the remaining 11, 904 genes (mostly low expressing genes) confirms an increase in drift-variance with age that is suppressed by mianserin (Figure 2—figure supplement 2g). To identify gene-sets that cannot possibly originate from the FUDR-treated eggs we exploited the specific arrest in embryonic development caused by FUDR. The DIC images suggested that FUDR arrests embryonic development before the birth of AFD, ASE and NSM neurons. If so, genes in our data-set that are specifically expressed in these neurons have to originate from the adult somatic tissue. To test that FUDR treatment prevents the birth of these neurons, we imaged eggs of C. elegans carrying a Pgcy-8::GFP transgene (AFD marker) (Figure 2—figure supplement 2h, i, j). Eggs from untreated animals showed a clear expression of the marker while FUDR-treated eggs did not (Figure 2—figure supplement 2i, j (n>100)). FUDR did not repress the expression of the Pgcy-8::GFP transgene in adults, showing that the lack of a Pgcy-8::GFP signal in FUDR-treated eggs is due to an arrest before the neurons are born and not due to inhibition of the reporter expression by FUDR. As AFD neurons are born before ASE and NSM neurons, these results suggested that none of these three neurons are present in FUDR-treated eggs (Sulston et al., 1983). After having established the absence of AFD, ASE and NSM neurons in eggs derived from FUDR treated animals, we then used the published gene-sets that are highly enriched in these three neuron types (AFD, ASE, NSM) to construct drift-plots (Etchberger et al., 2007; Spencer et al., 2014). Even for these highly restricted sets of genes, drift-variance dramatically increased with age and was repressed by mianserin. Taken together, these results show that the RNA contamination from FUDR-treated eggs is minimal and that this residual amount does not influence our results.