Imaging was done on a Bio-Rad Gel Doc XR+ imager using the default settings for GelRed with ultraviolet illumination. Images were typically taken at multiple exposures ranging from 5 to 45 s to facilitate accurate quantification. For each gel band, quantification was done using the highest-exposure image that did not contain saturated pixels in the band of interest (one exception—gel images for data shown in Fig. 2D required analysis of two partially saturated bands affecting two highest data points for the 15-min time). Thus, some gels were analyzed using multiple differently exposed images depending on the band brightness. These analyzed images were then normalized to the highest exposure. Nominally, this is done by scaling the ratio of the exposure times, but we additionally multiplied by a correction factor of 0.86 obtained by quantifying the same gel band across multiple identical images at different exposures. Efforts were made to take identical size images, but differently sized images could be normalized by the ratio of their pixel sizes. These normalizations only affected Fig. 2D, and the relative normalizations between small and total RNA are shown in Fig. 3, F and G.

To quantify each gel band, 12-bit images were imported into ImageJ and processed with a 2-pixel median filter to help reduce noise and small speckles and to eliminate hot pixels. Gel images were then rotated 90°, and a rectangular selection (of common size) was applied to each gel lane. In rare cases, where a bright speckle interfered with the band, we applied a smaller rectangle to analyze a fraction of the band. The plot profile command was used to get a mean intensity profile along the band width, and the area under the curve was quantified as a measure of the overall signal. While this analysis can also be done in ImageJ, we preferred to export the profiles to Origin 2017 (OriginLab) for a more precise quantification. In Origin, we used the peak analysis tool to draw a spline-interpolated baseline and measure the area between the profile and baseline. For the more complex multiplexing experiment, we instead explicitly subtracted the baseline using the same method and fit the subsequent profile with four Gaussian curves.

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