Capture RBP -RNA Complexes on Beads
Eric L. Van Nostrand, Thai B. Nguyen, Chelsea Gelboin-Burkhart, Ruth Wang, Steven M. Blue, Gabriel A. Pratt, Ashley L. Louie, Gene W. Yeo
Abstract
Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.
Steps
Couple Antibody to Magnetic Beads (Start During Cell Lysis)
Use 125µL + 10µg per sample.
Prewash Beads
Magnetically separate beads, remove the supernatant.
Wash beads 2× in 500µL.
Resuspend beads in 100µL .
Bind Antibody
Add 10µg to 100µL washed beads.
Rotate at Room temperature for 0h 45m 0s.
Capture RBP -RNA Complexes on Beads
Wash antibody beads 2× in 500µL.
Add the supernatant from Step 9 of Cell Lysis and Fragmentation to washed antibody beads.
Rotate at 4°C 0h 45m 0s.
Remove Input Samples
To a new tube, take 20µL for Preparative gel, store at 4°C.
To a new tube, take 20µL for Imaging gel, store at 4°C.
