Journal: PLoS ONE
Article Title: Analysis of the nucleocytoplasmic shuttling RNA-binding protein HNRNPU using optimized HITS-CLIP method
Figure Lengend Snippet: Schematic workflow of optimized CLIP. Cells are irradiated with 254 nm UV light on ice to form crosslinked RNA-RBP complexes, followed by partial RNase digestion and immunoprecipitation with RBP-specific antibodies. RNA crosslinked with RBPs is dephosphorylated for linker ligation, phosphorylated with [γ- 32 P]- ATP if necessary, separated by NuPAGE, and then transferred to a membrane. Isolated RNA is ligated with a 5'-App linker at the 3'-end and is subjected to BrdU-labeled cDNA synthesis by reverse transcription using a primer with a barcode and cleavage site. The cDNA is purified with the anti-BrdU antibody-coupled magnetic beads, circularized, digested with APE I, and amplified by PCR. Lastly, the CLIP cDNA library is subjected to size purification by PAGE. Steps modified from the original BrdU-CLIP protocol are indicated in red.
Article Snippet: The cDNA from 100 ng of total RNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions.
Techniques: Cross-linking Immunoprecipitation, Irradiation, Immunoprecipitation, Ligation, Isolation, Labeling, Purification, Magnetic Beads, Amplification, Polymerase Chain Reaction, cDNA Library Assay, Polyacrylamide Gel Electrophoresis, Modification