Probe hybridization to capture areas having a c18RNA footprint from 18S rRNA or printed control probe was done by pre-heating a hybridization mixture to 50 °C containing the following final concentrations at pH 8.0: 10 mM Tris–HCl (T6666, Sigma-Aldrich), 1 mM EDTA (108454, MERCK), 50 mM NaCl (S9888, Sigma-Aldrich), and 0.5 µM of fluorescently labeled probe (Supplementary Table 1 , IDT) and adding this mixture to each capture area in the hybridization cassette (AHC1X16, ArrayIT Corporation) for 10 min at RT. Then the mixture was pipetted off and the sRIN slide was removed from the hybridization cassette (AHC1X16, ArrayIT Corporation) and washed under continuous shaking at 300 rpm first in 2 × SSC (S6639, Sigma-Aldrich) with 0.1% SDS (71736, Sigma-Aldrich) at 50 °C for 10 min, followed by 0.2 × SSC (S6639, Sigma-Aldrich) at RT for 1 min, then in 0.1 × SSC (S6639, Sigma-Aldrich) at RT for 1 min. Finally, the sRIN slide was spin-dried.
Imaging was done using a DNA microarray scanner (InnoScan 910, Innopsys, France) with the following settings: excitation wavelength 532 nm set to same gain for all images (gain range: 20–70) and 635 nm set to gain 1. Then images were analyzed for FU using (Mapix, Innopsys, France).
Imaging was done using a DNA microarray scanner (InnoScan 910, Innopsys, France) with the following settings: excitation wavelength 532 nm set to same gain for all images (gain range: 20–70) and 635 nm set to gain 1. Then images were analyzed for FU using (Mapix, Innopsys, France).