Extended Data Fig. 1: Ribo-ITP design enables efficient RNA extraction and size selection.
From: Single-cell quantification of ribosome occupancy in early mouse development
a, The top view of the ITP chip layout designed with SOLIDWORKS (units in mm). The thickness of the channel features was 375 µm and that of the rectangular base was 1.5 mm. Linear tapering was applied from the rectangular base to the outer edge (rounded rectangle). b, Microfluidic device setup; indicating lysate, extraction, and size-selection channels; trailing electrolyte (TE) and leading electrolyte (LE) reservoirs; and elution well. Buffers corresponding to each channel and reservoir are color coded. Marker oligonucleotide fluorescence is denoted by green. c, Gel image displays the relative mobilities of fluorescent markers used in Ribo-ITP (M) along with the Zymo Research R1090 small RNA ladder (Z) and synthetic RNA oligonucleotides (S) (n = 1). d, Given that pH is the critical factor determining dephosphorylation efficiency, we determined the impact of Ribo-ITP collection on the conductivity and pH of the dephosphorylation buffer. We found negligible pH change (right axis, blue) and only a 11.0% ± 1.83% (SEM) change in conductivity (left axis, green) for the collection distance (5mm, denoted by the vertical line) used in Ribo-ITP. e, Representative gel images of control inputs (I), Ribo-ITP elutions (R), and gel extraction (G) samples. Four RNA species (17, 21, 25, and 29 nt) were used with total inputs of 20 and 40 ng. Fluorescent marker oligonucleotides were spiked into control and gel extraction samples prior to gel visualization. f, Gel image quantification of control inputs (gray), Ribo-ITP elutions (orange), and gel extraction (purple) samples (n = 4). Minimum, maximum, and average values are represented by the box and the horizontal bar. Only the 25 and 29 nt RNA marker bands were quantified for the yield calculation. g, Inputs (I, gray) were prepared by adding 40 ng of RNA to lysates from ~1,000 K562 cells. The RNA consisted of four species ranging from 17 to 29 nt in length. Fluorescent marker DNAs were added to Ribo-ITP samples (R) in addition to EGTA (10 mM). RNA extraction and isolation was done with Ribo-ITP followed by visualization using gel electrophoresis. h, Yield of the 25 and 29 nt RNAs was quantified and plotted for two replicates (84% and 91%).
