Dephosphorylation of 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
TAP Treat Input RNA
Prepare TAP master mix:
| A | B |
|---|---|
| H2O | 10 μL |
| 10× TAP buffer | 2.5 μL |
| RNase Inhibitor | 0.5 μL |
| TAP enzyme | 2.5 μL |
To INPUT samples ONLY , add 15.5µL .
Mix and incubate in Thermomixer at 1200rpm,37°C.
PNK Treat Input RNA
Prepare PNK master mix (75 μL per sample):
| A | B |
|---|---|
| H2O | 45 μL |
| 5× PNK pH 6.5 buffer | 20 μL |
| 0.1 M DTT | 1 μL |
| DNase | 1 μL |
| Murine RNase Inhibitor | 1 μL |
| T4 PNK enzyme | 7 μL |
Add 75µL to samples and mix. Incubate in Thermomixer at 1200rpm,37°C.
Silane Cleanup Input RNA
Prepare beads:
Magnetically separate 20µL per sample, remove the supernatant.
Wash 1× with 900µL .
Resuspend beads in 300µL per sample.
Bind RNA:
Add beads in 300µL to sample, mix.
Add 10µL .
Add 615µL .
Mix, rotate at Room temperature, 0h 15m 0s.
Wash beads:
Magnetically separate, remove the supernatant.
Add 1mL, pipette resuspend and move suspension to 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 and transfer 5µL to a new tube (for 3′ RNA adapter ligation). Transfer the remainder of the supernatant to a new tube and store at -20°C (as a backup input RNA sample).
