Fig. 1: A method for determination of RBP-RNA interaction in vivo in mice.

a Schematic representation of in vivo PAR-CLIP (viP-CLIP) applied in mice. Mice are injected 6 times with 4SU over the time course of 12.5 h and sacrificed 15 h after the first dose. Intact organs are flash-frozen, grinded, and cross-linked with UV light (365 nm) under liquid nitrogen cooling. After cell lysis of the tissue powder, the RNA-RBP complexes are immunoprecipitated in presence of RNase, radiolabeled and the recovered RNA in the size of 19–35 nt is ligated to sequencing adapter, and used for cDNA library preparation and sequencing. b 4SU incorporation rates in tissues (n = 3) after injection regime displayed in (a). HEK293 (n = 3) and primary hepatocytes (n = 3) were cultured in the presence of 100 mM 4SU for 16 h and subjected together with the tissue samples to HPLC analysis of 4SU incorporation rates. Values are mean +/– S.D. c Phosphorimage of urea-PAGE transferred to a nitrocellulose membrane that resolved ³²P-labeled RNA-TIAL1 complexes after immunoprecipitation of TIAL1 in several tissues. d Immunoblot showing tissue distribution of TIAL1 in mouse organs. e Autoradiograph of recovered RNAs as displayed in (c), after proteinase K treatment and 15% urea gel electrophoresis. 5’ radiolabeled synthetic RNAs of 19 and 35 nt length served as size markers. c, d, e Representative images of three independent replicates. f Graphic presentation of viP-CLIP cDNA library composition by RNA categories from recovered RNA from (e). Source data are provided as a Source Data file.