3' RNA Adapter Ligation to Input RNA
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
Anneal Adapter
Take 5µL (from Dephosphorylation of Input RNA).
Add 1.5µL and 0.5µL ( see Note 3 3).
Incubate at 65°C for 0h 2m 0s. Place On ice > 0h 1m 0s.
Prepare Ligation Master Mix; 13.5 μL Per Sample
| A | B |
|---|---|
| 10× Ligase Buffer (with DTT) | 2.0 μL |
| 0.1 M ATP | 0.2 μL |
| Murine RNase Inhibitor | 0.2 μL |
| 100% DMSO | 0.3 μL |
| 50% PEG 8000 | 8.0 μL |
| RNA Ligase high conc | 1.3 μL |
| H2O | 1.5 μL |
Add 13.5µL to each sample, mix, incubate at Room temperature for 1h 15m 0s. Flick to mix every ~0h 15m 0s.
Silane Cleanup Input RNA
Prepare beads:
Magnetically separate 20µL per sample, remove the supernatant.
Wash 1× with 900µL and resuspend beads in 61.6µL .
Bind RNA:
Add beads in 61.6µL to sample, mix and add 61.6µL .
Pipette mix, leave pipette tip in a tube, pipette mix every ~ 0h 3m 0s –5 min for 0h 15m 0s.
Wash beads:
Magnetically separate, remove the supernatant.
Add 1mL, pipette resuspend and move to a new tube.
After 0h 0m 30s, magnetically separate, remove the supernatant, and wash 2× with 75% EtOH (0h 0m 30s).
Magnetically separate, remove residual liquid with fine tip → Air-dry 0h 5m 0s.
Elute RNA:
Resuspend in 10µL, let it sit for 0h 5m 0s.
Magnetically separate, transfer the supernatant to a new tube.
