Figure 2.

Quantification of Y RNA levels. Human Y RNAs were synthesized by in vitro transcription. Cellular total RNA was extracted from HEK293 cells using TRIZOL (Sigma-Aldrich). The amount of cellular RNA was normalized to the cell number and corrected for purification efficiencies. Then quantitative northern blotting (Li-COR imaging system) was performed to determine the number of Y RNA molecules in HEK293 cells. Each Y RNA was quantified using in vitro transcribed RNA as reference. Y RNA quantification was repeated three times and cellular Y RNA copies were calculated resulting in 12 data points per Y RNA. The results of 12 quantifications are shown on the left; the mean number of cellular Y RNAs is depicted on the right. Representative images of the resulting northern blots are depicted in Supplementary Figure S1. The in vitro transcription procedure as well as the used northern blotting protocols were described previously (Kohn et al., 2015).