3' Linker Ligate cDNA (On-Bead), and 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 Linker
Add 0.8µL ( see Note 3 3).
Add 1µL .
Heat at 75°C, 0h 2m 0s, place immediately On ice for > 0h 1m 0s.
Prepare Ligation Master Mix on Ice; 12.8 μL Per Sample
Prepare Ligation Master Mix on Ice; 12.8 μL Per Sample:
| A | B |
|---|---|
| 10× RNA Ligase Buffer (with DTT) | 2.0 μL |
| 0.1 M ATP | 0.2 μL |
| 50% PEG 800 | 9.0 μL |
| High concentration T4 RNA Ligase | 0.5 μL |
| H2O | 1.1 μL |
Flick to mix, spin down, and add 12.8µL to each sample: add master mix slowly with stirring; it needs to be homogeneous.
Add an additional 1µL on the top of sample and pipette mix.
Incubate at Room temperature 0h 1m 0s.
Silane Cleanup Linker-Ligated cDNA
Prepare beads:
Magnetically separate 5µL per sample, remove the supernatant.
Wash 1× with 500µL.
Resuspend beads in 60µL per sample.
Bind RNA:
Add beads in 60µL to each sample, mix and add 60µL.
Pipette mix, incubate for 0h 5m 0s at Room temperature.
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.
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 27µL, let it sit for 0h 5m 0s.
Magnetically separate, transfer 25µL to a new tube.
