TITLE: Immunoprecipitation Washes and RNA Dephosphorylation
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]
?. [Wash Beads (Pre-chill All Wash Buffers to 4 °C)]
Magnetically separate beads, remove the supernatant (or store for western if desired).
We often perform intermediate washes with equal mixtures of the previous and current wash buffer, in order to decrease dramatic changes in buffer composition.
?. [Wash Beads (Pre-chill All Wash Buffers to 4 °C)]
Wash 2× with , then 1× with . Remove the supernatant.
[High salt wash buffer]
[Wash buffer]
?. [Wash Beads (Pre-chill All Wash Buffers to 4 °C)]
Add , mix, separate on a magnet, add , mix, remove the supernatant. Wash 1× with , remove the supernatant.
[Wash buffer]
[1× TAP buffer]
[1× TAP buffer]
?. [TAP Treat (on-Bead)]
Prepare TAP master mix  ( per sample):  AB1H2O79 μL210× TAP buffer10 μL3Murine RNase Inhibitor2 μL4DNase1 μL5TAP enzyme8 μL
on ice
100 µl
AB1H2O79 μL210× TAP buffer10 μL3Murine RNase Inhibitor2 μL4DNase1 μL5TAP enzyme8 μL
?. [TAP Treat (on-Bead)]
Add  to each sample, incubate in Thermomixer at .
[TAP mix]
37 33
?. [PNK Treat (On-Bead)]
Prepare PNK master mix  ( per sample):   AB1H2O224 μL25× PNK pH 6.5 buffer60 μL30.1 M DTT3 μL4Murine RNase Inhibitor5 μL5DNase1 μL6T4 PNK enzyme7 μL
on ice
300 µl
AB1H2O224 μL25× PNK pH 6.5 buffer60 μL30.1 M DTT3 μL4Murine RNase Inhibitor5 μL5DNase1 μL6T4 PNK enzyme7 μL
?. [PNK Treat (On-Bead)]
Add  to each sample, incubate in Thermomixer at .
[PNK mix]
100 33
?. [Wash Beads (Prechill Buffers to 4 °C)]
Magnetically separate beads, remove the supernatant.
?. [Wash Beads (Prechill Buffers to 4 °C)]
Wash 1× with , remove the supernatant.
[Wash buffer]
?. [Wash Beads (Prechill Buffers to 4 °C)]
Add , mix, add , mix, remove the supernatant.
[Wash buffer]
[High salt wash buffer]
?. [Wash Beads (Prechill Buffers to 4 °C)]
Add , mix, add , mix, remove the supernatant.
[High salt wash buffer]
[Wash buffer]
?. [Wash Beads (Prechill Buffers to 4 °C)]
Wash 1× with , remove the supernatant.
[Wash buffer]
?. [Wash Beads (Prechill Buffers to 4 °C)]
Add , mix, add , mix, remove the supernatant.
[Wash buffer]
[1× RNA Ligase buffer (no DTT)]
?. [Wash Beads (Prechill Buffers to 4 °C)]
Repeat wash 2× with , carefully remove all remaining supernatant.
[1× RNA Ligase buffer (no DTT)]