Non-poly(A) RNA Interactome capture (RIC) of mouse liver

Male mice on a homogenous C57BL6/J genetic background were sacrificed at 11 to 13 weeks of age by cervical dislocation. Liver were immediately harvested, flash frozen in liquid nitrogen and stored at -80°C until use. Animal handling was in accordance with guidelines approved by the European Molecular Biology Laboratory (EMBL). Organ sections (thickness 30 µm) were prepared in a Cryostat (Leica Biosystems, Leica CM3050 S) set to -20 °C and deposited onto SuperFrost glass slides (Carl Roth, H880); the glass slides were pre-cooled to -20°C in the cryostat. ~10 sections were placed on a glass slide, and a total of around 150 sections were prepared for each sample. Tissue sections on glass slides were then transferred onto metal plates placed on dry ice. The tissue sections were exposed or not to 1 J/cm2 of 254 nm UV light in a XL 1500 UV Spectrolinker (Spectronics Corporation) and subjected to eRIC (PMID: 30352994). Poly(A) RNA-depleted supernatants obtained after two consecutive rounds of eRIC were stored at -80ºC and then used as input for the column-based guanidinium thiocyanate-phenol-chloroform extraction of ribonuleoprotein (RNP) complexes, applying an adaptation of the 2C method (PMID: 30035255). Four independent irradiated samples were employed. For the non-irradiated control, organ sections from four mice were pooled to generate one non-crosslink sample. 40 µg of captured RNA per sample were employed.