Reverse Transcribe RNA (All Clip and Input Samples), and Reaction Cleanup
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 Primer in 8-Well Strip Tubes
Mix 10µL with 0.5µL.
Heat at 65°C for 0h 2m 0s in preheated PCR block, place immediately On ice.
Prepare Reverse Transcription Master Mix on Ice; 10 μL Per Sample
| A | B |
|---|---|
| H2O | 4.0 μL |
| 10× AffinityScript Buffer | 2.0 μL |
| 0.1 M DTT | 2.0 μL |
| dNTPs (100 mM; 25 mM each) | 0.8 μL |
| Murine RNase Inhibitor | 0.3 μL |
| AffinityScript Enzyme | 0.9 μL |
Add 10µL to each sample, mix, and incubate at 55°C for 0h 45m 0s in preheated PCR block.
Cleanup cDNA
Removal of excess primers
Add 3.5µL to each sample, vortex, spin down.
Incubate at 37°C for 0h 15m 0s on a PCR block.
Add 1µL, pipette-mix.
RNA removal:
Add 3µL, pipette-mix.
Incubate at 70°C for 0h 12m 0s on a PCR block.
Add 3µL, pipette-mix.
Silane Cleanup cDNA
Prepare beads:
Magnetically separate 10µL per sample, remove the supernatant.
Wash 1× with 500µL and resuspend beads in . 93µL.
Bind RNA:
Add beads in 93µL to sample, mix and add 111.6µL .
Pipette mix, leave pipette tip in a tube, pipette mix twice, for 0h 5m 0s.
Wash beads:
Magnetically separate, remove the supernatant.
Add 1mL, pipette resuspend and move to new tube.
After 0h 0m 30s, magnetically separate, remove the supernatant, and wash 2× with 80% EtOH (0h 0m 30s).
Magnetically separate, remove residual liquid with fine tip → air-dry 0h 5m 0s.
Elute RNA:
Resuspend in 5µL, let it sit for 0h 5m 0s (do NOT remove from beads).
