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Using Cross-Linking and ImmunoPrecipitation (CLIP) to Identify Targets and Specificity Determinants of the YTHDC2
Yuhki Saito1,2, Devanshi Jain3, Robert B Darnell4
1Laboratory of Molecular Neuro-oncology, Howard Hughes Medical Institute, Rockefeller University, New York, NY, USA. yuki.saito.01@daiichisankyo.com.
Abstract:
The 3'→5' RNA helicase YTHDC2 is a posttranscriptional regulator of gene expression in reproductive cells, where it is essential for the progression of meiosis. But a detailed understanding of its molecular mechanism is currently lacking. Cross-linking and immunoprecipitation (CLIP) followed by high-throughput sequencing is a well-established method for transcriptome-wide identification of the positions of protein-RNA interactions and has played a critical role in studies of RNA-binding proteins. In this chapter, we present an optimized protocol for performing YTHDC2 CLIP in mouse testes to identify YTHDC2-RNA interactions, from harvesting testis tissue, through isolation of cross-linked YTHDC2-RNA complexes, to the preparation of sequencing libraries.