Supplementary Figure 5: Effect of s4U treatment on mESC viability and metabolic RNA labeling.
From: Thiol-linked alkylation of RNA to assess expression dynamics
(a) Viability of mESCs cultured in the presence of the indicated concentration of 4-thiouridine (s4U) for 12 h (left) or 24 h (right) relative to untreated conditions is shown. Note, that s4U-containing media was exchanged every 3h in the course of the labeling experiment. Final concentration used in subsequent experiments (100 μM) is indicated by triangle and dotted line. Mean ± SD of three independent cell cultures are shown. (b) Quantification of s4U-incorporation into total RNA after s4U-metabolic labeling incubated for the indicated time in a pulse, or following media replacement after 24 h pulse labeling followed by uridine-chase for the indicated times. s4U-incorporation was determined by HPLC analysis following digestion and dephosphorylation of total RNA to single nucleosides. Representative absorbance spectrum of background-subtracted s4U signal intensities normalized to 24 h pulse labeling time point is shown. Column retention time (min) relative to s4U-adsorption maxima are shown. (c) Substitution rate of s4U compared to unmodified uridine determined by HPLC as shown in (b) and previously described (Spitzer et al., 2014). s4U incorporation in total RNA across all time points of a s4U-metablic pulse and chase labeling experiment in mESCs. Values represent mean ± SD of three independent cell cultures. Maximum incorporation rates after 24 h labeling are indicated. (Note, that median s4U incorporation frequencies for mRNA [Fig. 2c] are higher than estimated by HPLC analysis of single nucleoside- digested total RNA, most certainly because stable RNA polymerase I and III transcripts, such as rRNA and tRNA, are strongly overrepresented in total RNA but depleted from RNA polymerase II-specific Quant-seq libraries. See also Supplementary Fig. 9.)
Spitzer, J. et al. PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation): a step-by-step protocol to the transcriptome-wide identification of binding sites of RNA-binding proteins. Meth Enzymol 539, 113–161 (2014).
