TITLE: Dephosphorylation of Input RNA
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]
?. [TAP Treat Input RNA]
Prepare TAP master mix:  AB1H2O10 μL210× TAP buffer2.5 μL3RNase Inhibitor0.5 μL4TAP enzyme2.5 μL
AB1H2O10 μL210× TAP buffer2.5 μL3RNase Inhibitor0.5 μL4TAP enzyme2.5 μL
?. [TAP Treat Input RNA]
To INPUT samples ONLY, add .
[TAP master mix]
?. [TAP Treat Input RNA]
Mix and incubate in Thermomixer at .
Centrifuge: 1200 33, 15 min, 37 10
?. [PNK Treat Input RNA]
Prepare PNK master mix (75 μL per sample):  AB1H2O45 μL25× PNK pH 6.5 buffer20 μL30.1 M DTT1 μL4DNase1 μL5Murine RNase Inhibitor1 μL6T4 PNK enzyme7 μL
AB1H2O45 μL25× PNK pH 6.5 buffer20 μL30.1 M DTT1 μL4DNase1 μL5Murine RNase Inhibitor1 μL6T4 PNK enzyme7 μL
?. [PNK Treat Input RNA]
Add  to samples and mix. Incubate in Thermomixer at .
[PNK master mix]
Centrifuge: 1200 33, 20 min, 37 10
?. [Silane Cleanup Input RNA]
Prepare beads:
?. [Silane Cleanup Input RNA]
Magnetically separate  per sample, remove the supernatant.
[MyONE Silane beads]
?. [Silane Cleanup Input RNA]
Wash 1× with .
[RLT buffer]
?. [Silane Cleanup Input RNA]
Resuspend beads in  per sample.
[RLT buffer]
?. [Silane Cleanup Input RNA]
Bind RNA:
?. [Silane Cleanup Input RNA]
Add beads in  to sample, mix.
[RLT buffer]
?. [Silane Cleanup Input RNA]
Add .
[5 M NaCl]
?. [Silane Cleanup Input RNA]
Add .
[100% EtOH]
?. [Silane Cleanup Input RNA]
Mix, rotate at , .
0 Room temperature
?. [Silane Cleanup Input RNA]
Wash beads:
?. [Silane Cleanup Input RNA]
Magnetically separate, remove the supernatant.
?. [Silane Cleanup Input RNA]
Add , pipette resuspend and move suspension to new tube.
[75% EtOH]
?. [Silane Cleanup Input RNA]
After , magnetically separate, remove the supernatant, and wash 2× with 75% EtOH ().
?. [Silane Cleanup Input RNA]
Magnetically separate, remove residual liquid with fine tip → air-dry .
?. [Silane Cleanup Input RNA]
Elute RNA:
?. [Silane Cleanup Input RNA]
Resuspend in , let it sit for .
[ H2O]
?. [Silane Cleanup Input RNA]
Magnetically separate and transfer  to a new tube (for 3′ RNA adapter ligation). Transfer the remainder of the supernatant to a new tube and store at  (as a backup input RNA sample).
[supernatant]
-20 °C