Cross-linking immunoprecipitation (CLIP). Tissue (e.g., brain) or cells are UV-irradiated (see Protocol: Ultraviolet (UV) Cross-Linking of Live Cells, Lysate Preparation, and RNase Titration for Cross-Linking Immunoprecipitation (CLIP) [Darnell et al. 2018a]), inducing covalent cross-links between RNA–protein complexes in vivo. (Arrow 1, see Protocol: Ultraviolet (UV) Cross-Linking of Live Cells, Lysate Preparation, and RNase Titration for Cross-Linking Immunoprecipitation (CLIP) [Darnell et al. 2018a] and Protocol: Immunoprecipitation and SDS-PAGE for Cross-Linking Immunoprecipitation (CLIP) [Darnell et al. 2018b]) Cell lysis and partial RNase digestion allow partial clarification of RNA–protein complexes before immunoprecipitation and reduce the modal size of cross-linked RNA to a size determined by the experimenter (in typical experiments, this would be ∼50 nt or smaller). RNase A and T1 leave a 5′-OH and a 3′-phosphate group on the digested RNA. (Arrow 2, see Protocol: Immunoprecipitation and SDS-PAGE for CLIP [Darnell et al. 2018b]) Cross-linking allows stringent conditions to be used for protein purification. Immunopurification using antibodies against native epitopes or protein tags can be used. (Arrows 3, 4, see Protocol: 3′-Linker Ligation and Size Selection by SDS-PAGE for Cross-Linking Immunoprecipitation (CLIP) [Darnell et al. 2018c]) The 3′ phosphate is removed by alkaline phosphatase, to prevent intramolecular RNA circularization during ligation of a linker to the 3′ end of the RNA. This linker is itself blocked at the 3′ end with a puromycin moiety to prevent competing linker–linker ligation reactions. (Arrow 5, see Protocol: 3′-Linker Ligation and Size Selection by SDS-PAGE for Cross-Linking Immunoprecipitation (CLIP) [Darnell et al. 2018c]) RNA is labeled at the 5′ end with T4 polynucleotide kinase and [γ-³²P]ATP. (Arrow 6, see Protocol: 3′-Linker Ligation and Size Selection by SDS-PAGE for (CLIP) [Darnell et al. 2018c]) RNABP:RNA complexes are released from beads, run on denaturing SDS-PAGE and transferred to nitrocellulose (two important purification steps), and imaged by autoradiography. (Arrow 7, see Protocol: Isolation of the RNA Cross-Linking Immunoprecipitation (CLIP) Tags, 5′-Linker Ligation, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Amplification, and Sequencing [Darnell et al. 2018d]) The radioactive RNABP–protein complex is excised from the nitrocellulose filter and digested with proteinase K to remove the RNABP and elute the RNA, which is then isolated by phenol:chloroform extraction and ethanol precipitation. (Arrow 8, see Protocol: Isolation of the RNA Cross-Linking Immunoprecipitation (CLIP) Tags, 5′-Linker Ligation, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Amplification, and Sequencing [Darnell et al. 2018d]) A second linker is added to the 5′ end of the RNA. (Arrow 9, see Protocol: Isolation of the RNA Cross-Linking Immunoprecipitation (CLIP) Tags, 5′-Linker Ligation, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Amplification, and Sequencing [Darnell et al. 2018d]) The RNA is amplified by RT-PCR and sequenced.