TITLE: Cell Lysis and RNA Fragmentation
AUTHORS: Eric L. Van Nostrand, Thai B. Nguyen, Chelsea Gelboin-Burkhart, Ruth Wang, Steven M. Blue, Gabriel A. Pratt, Ashley L. Louie, Gene W. Yeo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [Lyse Cells]
Add   +  to each pellet.
[cold lysis buffer]
[200× Protease Inhibitor Cocktail III]
RNase inhibitor addition to lysis buffer.Murine RNase inhibitor (NEB) inhibits many endogenous RNase enzymes but does not significantly inhibit RNase I. As such, it can be added either before or after RNase I treatment. For many cell lines (HEK293T, K562) we have observed that this choice does not alter fragmentation or ultimate signal. However, we have observed that for cell types or tissues with moderate to high endogenous RNase activity (e.g., stem cells, differentiated neurons, many tissues), the addition of RNase inhibitor at the initial lysis step is essential to prevent over-fragmentation. We note that the amount of RNase inhibitor may need to be further increased for samples with particularly high RNase activity (e.g., liver or pancreas).
?. [Lyse Cells]
Pipette to resuspend, incubate for .At this time, begin antibody coupling (Capture RBP -RNA Complexes on Beads).
on ice
?. [RNase Treat Lysate]
Sonicate in Bioruptor at “low” setting, 30 s on / 30 s off for  at .
4 °C
?. [RNase Treat Lysate]
Add  to lysate.
[DNase ]
?. [RNase Treat Lysate]
Dilute RNase I 1:25 in 1× PBS (prechilled to ).
4 °C
?. [RNase Treat Lysate]
Add  to lysate, mix and immediately proceed to the next step.
[diluted RNase I ]
?. [RNase Treat Lysate]
Incubate in Thermomixer at .
Centrifuge: 1200 33, 5 min, 37 10
?. [RNase Treat Lysate]
Place  , add  and pipette mix.
on ice
[Murine RNase Inhibitor]
RNase inhibitor addition to lysis buffer.Murine RNase inhibitor (NEB) inhibits many endogenous RNase enzymes but does not significantly inhibit RNase I. As such, it can be added either before or after RNase I treatment. For many cell lines (HEK293T, K562) we have observed that this choice does not alter fragmentation or ultimate signal. However, we have observed that for cell types or tissues with moderate to high endogenous RNase activity (e.g., stem cells, differentiated neurons, many tissues), the addition of RNase inhibitor at the initial lysis step is essential to prevent over-fragmentation. We note that the amount of RNase inhibitor may need to be further increased for samples with particularly high RNase activity (e.g., liver or pancreas).
?. [RNase Treat Lysate]
Centrifuge  → Transfer the supernatant to a new tube and discard pellet.
Centrifuge: 15000 34, 15 min, 4 10