Protocol for quantifying silent ribosome induction in yeast and mammalian cells using polysome profiling

Figures

• 

  Figure 1. Scheme for RNA extraction from polysome profile fractions The fractions generated from the polysome profile were first pooled as shown and a unit volume of spike-in RNAs was added to each pool. The pools were then diluted into multiple tubes using the considerations explained in steps 15–17. Following this, the RNA was cleaned up by phenol-chloroform and ethanol precipitation and then the diluted tubes from each pool were merged. Finally, a second, column-based cleanup was carried out for each pool.

• 

  Figure 2. Scheme for quantifying silent ribosome induction by qPCR Scheme shows an outline of the processes detailed in step 31.a.

• 

  Figure 3. Scheme for quantifying silent ribosome induction by RNA-seq Scheme shows an outline of the processes detailed in step 31.b.

• 

  Figure 4. Dilution of the polysome profiling fractions leads to consistent RNA extraction (A) Comparison of two similar polysome profiling experiments which differed only in the fact that in one, the fractions were pooled and diluted as described in this paper and in the other, they were extracted without dilution. We see that in the non-diluted sample there is a greater variation in the spike-in intensities and especially the heavier polysomes which would have higher sucrose yield lesser RNA. (B) The mean CV of the spike-in RNAs across all pools/fractions, calculated for two different spike-ins, for the experiments in which the fractions were diluted (red) and non-diluted (blue). Error bars show standard error of the mean (SEM), N=2 replicates.

• 

  Figure 5. Polysome profile under standard and stress conditions Polysome profile traces of BY4743 cells under standard conditions (2% glucose, black solid line), ethanol stress (12% EtOH, 20 min, red dashed line), and glucose depletion stress (0% glucose, 20 min, blue dashed line).

• 

  Figure 6. Consistency of the 5S/28S (Rn5S/Rn28S1) rRNA ratios measured with RNA-seq in macrophages No additional column-based purification was performed to remove 5S rRNA. The non-depleted RNA samples isolated from the polysome profiling fractions are shown at the indicated ribosome numbers. The peak values of the bell-shaped curves denote the ratio. Bell-shaped curves are schematic and serve only as positional markers.

• 

  Figure 7. Estimation of the silent ribosomes using RNA-seq in macrophages RNA-seq data obtained from RNA samples in which rRNA was not depleted. mRNAs and rRNAs (5.8S, 18S, and 25S) levels were summed in TPM units to calculate the ∑mRNA/∑(rRNA-{5S}) ratio. The peak values of the bell-shaped curves denote the ratios at 37°C (control) and 42°C (heat stress). Bell-shaped curves are schematic and serve only as positional markers.