Non-poly(A) RNA Interactome Capture (RIC) of mouse brain, kidney and liver

ABSTRACT: Male mice on a homogenous C57BL6/J genetic background were sacrificed at 11 to 13 weeks of age by cervical dislocation. Brain, kidneys and 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 per mouse. 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). Eluates obtained from UV-treated samples from the respective organs of two mice were combined per independent experiment, and the analyses were conducted in duplicate for each organ studied. For the non-crosslink controls, organ sections from four mice were pooled to generate one non-crosslink sample per organ studied. 2 µg of captured RNA per sample were employed.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain, Liver, Kidney

SUBMITTER: Frank Stein  

LAB HEAD: Matthias W. Hentze

PROVIDER: PXD038076 | Pride | 2023-03-02

REPOSITORIES: Pride