Fig. 3: A method to quantify the absolute number of ligated RNA–protein complexes.

a Fraction of hnRNP C UV cross-linked to RNA, including hnRNP C that cannot be collapsed to a monomeric band by RNAse. The experiment was performed twice with similar results. b Ligation of the L5 adapter creates a novel band of monomeric hnRNP C-RNA-L5. The experiment was performed three times with similar results. c Absolute quantification of the monomeric hnRNP C and L5-hnRNP C band using a standard curve of GST-hnRNP C quantifying the number of L5 molecules in the complex and thereby fluorescence per molecule. Black dots comprise the standard curve and red dots endogenous hnRNP C. d Schematic of the method to estimate the amount of fluorescence per L5 molecule lost in the CLIP procedure. e RNA purified as in d may be RNAse treated to collapse signal into adapter bands. The sizes of oligos are approximated with the dye molecule counting as 6 nt. The experiment was performed once. f The method in d applied to known amounts of free L5 and L3 adapter; note that adapters are completely shifted. The experiment was performed three times with similar results. g Application of the method in d to visualize fluorescence of hnRNP C-cross-linked RNA after a CLIP procedure. Visualization was performed once, not including the quantified samples below. h Fluorescence loss of L5 from the CLIP procedure in replicate experiments (n = 3). The box limits denote the 25–75% quartile range, the center line is the median, and whiskers show extreme values.