Crosslinking of Cultured Cells
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
Preparation of Cultured Cells for Crosslinking
Aspirate the spent media and wash the plate with 1× DPBS (~15mL for a 15 cm plate) at Room temperature. For suspension cells, pellet cells to remove spent media, and wash once with 1× DPBS.
Aspirate the supernatant from previous step, and add enough 1× DPBS (~5mL for a 15 cm plate) to cover the plate. For suspension cells, resuspend in 1× DPBS to a cell density of 2 × 107cells per mL, place 3mL of cell suspension in 1× DPBS on a 10 cm plate.
Crosslinking of Cultured Cells
Place the tissue culture plate On ice or cooling block, and place the entire apparatus into the UV crosslinker, making sure the plate is leveled. Remove lid prior to crosslinking.
Crosslink the cells with an energy setting of 400 mJ/Cm2.
While keeping the cells On ice, use a cell scraper to scrape the plate. Transfer the cells to a 50 mL conical tube. Wash the plate with 10mL and add the wash to the 50 mL conical tube. Gently resuspend the cell until a homogenous mixture is obtained.
Spin cells down at 200x g.
Aspirate the supernatant. Resuspend in the desired amount for flash freezing, typically 2 × 107cells per mL.
Transfer desired amount into 1.5 mL epi-tubes, and then spin down at 200x g. Aspirate the supernatant and freeze by submerging the epi-tubes completely in liquid nitrogen. Store at -80°C.
