Agarose Gel Electrophoresis and Size-Selection of Library
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
Prepare 3% low-melting temp agarose gel with 1:10,000 SybrSafe in 1× TBE.
Prepare Samples and Run Gel
Add 6µL to each sample (18 μL of sample), mix.
Load on gel, leave one empty well between samples, 50 bp ladder on both sides of the gel.
Run ~95 V for 0h 50m 0s (longer gives better resolution but larger cut sizes).
Gel-Extract Library from Gel
Under blue light illumination, cut gel slice from 175–350 bp and place into 15 mL conical tube.
Use fresh razor blades for each sample and keep cross-contamination to minimum.
Cut and elute gel using Qiagen MinElute gel extraction kit:
Add 6× volumes of Buffer QG to melt gel (e.g., for 100 mg gel, add 600 μL QG).
Melt gel at Room temperature (do not heat) on benchtop (can shake to help melt, but do not vortex).
Add 1× volume of original gel of isopropanol and mix well (100 mg gel = 100 μL isopropanol).
Load on column (750 μL per spin, can do multiple spins, all spins max speed 1 min).
After all sample has been spun through, wash 1× with 500µL .
Add 1× with 750µL, spin 0h 1m 0s, pour out flow-through, spin again 0h 2m 0s max speed.
Carefully move column to a new 1.5 mL tube, and air dry for 0h 2m 0s.
Carefully add 12.5µL directly to the center of the column, incubate for 0h 2m 0s at Room temperature, spin at max speed.
