TITLE: 3&#39; Linker Ligate cDNA (On-Bead), and Cleanup
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]
?. [Anneal Linker]
Add  (see Note 3).
[InvRand3Tr3 adapter ]
?. [Anneal Linker]
Add .
[100% DMSO]
?. [Anneal Linker]
Heat at , , place immediately  for >.
75 °C
on ice
?. [Prepare Ligation Master Mix on Ice; 12.8 μL Per Sample]
Prepare Ligation Master Mix on Ice; 12.8 μL Per Sample:  AB110× RNA Ligase Buffer (with DTT)2.0 μL20.1 M ATP0.2 μL350% PEG 8009.0 μL4High concentration T4 RNA Ligase0.5 μL5H2O1.1 μL
AB110× RNA Ligase Buffer (with DTT)2.0 μL20.1 M ATP0.2 μL350% PEG 8009.0 μL4High concentration T4 RNA Ligase0.5 μL5H2O1.1 μL
?. [Prepare Ligation Master Mix on Ice; 12.8 μL Per Sample]
Flick to mix, spin down, and add  to each sample: add master mix slowly with stirring; it needs to be homogeneous.
[ Ligation Master Mix]
?. [Prepare Ligation Master Mix on Ice; 12.8 μL Per Sample]
Add an additional  on the top of sample and pipette mix.
[High concentration T4 RNA Ligase]
?. [Prepare Ligation Master Mix on Ice; 12.8 μL Per Sample]
Incubate at .
0 Room temperature
?. [Silane Cleanup Linker-Ligated cDNA]
Prepare beads:
?. [Silane Cleanup Linker-Ligated cDNA]
Magnetically separate  per sample, remove the supernatant.
[MyONE Silane beads]
?. [Silane Cleanup Linker-Ligated cDNA]
Wash 1× with .
[RLT buffer]
?. [Silane Cleanup Linker-Ligated cDNA]
Resuspend beads in  per sample.
[RLT buffer]
?. [Silane Cleanup Linker-Ligated cDNA]
Bind RNA:
?. [Silane Cleanup Linker-Ligated cDNA]
Add beads in  to each sample, mix and add .
[RLT buffer]
[100% EtOH]
?. [Silane Cleanup Linker-Ligated cDNA]
Pipette mix, incubate for  at .
[(pipette mix twice with same tips during incubation)]
0 Room temperature
?. [Silane Cleanup Linker-Ligated cDNA]
Wash beads:
?. [Silane Cleanup Linker-Ligated cDNA]
Magnetically separate, remove the supernatant.
?. [Silane Cleanup Linker-Ligated cDNA]
Add , pipette resuspend and move to a new tube.
[75% EtOH]
?. [Silane Cleanup Linker-Ligated cDNA]
After , magnetically separate, remove the supernatant.
?. [Silane Cleanup Linker-Ligated cDNA]
Wash 2× with 75% EtOH ().
?. [Silane Cleanup Linker-Ligated cDNA]
Magnetically separate, remove residual liquid with fine tip → Air-dry .
?. [Silane Cleanup Linker-Ligated cDNA]
Elute RNA:
?. [Silane Cleanup Linker-Ligated cDNA]
Resuspend in , let it sit for .
[10 mM Tris–HCl pH 7.5]
?. [Silane Cleanup Linker-Ligated cDNA]
Magnetically separate, transfer  to a new tube.
[sample]