TITLE: Reverse Transcribe RNA (All Clip and Input Samples), and Reaction 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 Primer in 8-Well Strip Tubes]
Mix  with .
[RNA]
[InvAR17 primer]
?. [Anneal Primer in 8-Well Strip Tubes]
Heat at  for  in preheated PCR block, place immediately .
65 °C
on ice
?. [Prepare Reverse Transcription Master Mix on Ice; 10 μL Per Sample]
AB1H2O4.0 μL210× AffinityScript Buffer2.0 μL30.1 M DTT2.0 μL4dNTPs (100 mM; 25 mM each)0.8 μL5Murine RNase Inhibitor0.3 μL6AffinityScript Enzyme0.9 μL
AB1H2O4.0 μL210× AffinityScript Buffer2.0 μL30.1 M DTT2.0 μL4dNTPs (100 mM; 25 mM each)0.8 μL5Murine RNase Inhibitor0.3 μL6AffinityScript Enzyme0.9 μL
?. [Prepare Reverse Transcription Master Mix on Ice; 10 μL Per Sample]
Add  to each sample, mix, and incubate at  for  in preheated PCR block.
[Reverse Transcription Master Mix]
55 °C
?. [Cleanup cDNA]
Removal of excess primers
?. [Cleanup cDNA]
Add  to each sample, vortex, spin down.
[ExoSAP-IT]
?. [Cleanup cDNA]
Incubate at  for  on a PCR block.
37 °C
?. [Cleanup cDNA]
Add , pipette-mix.
[0.5 M EDTA]
?. [Cleanup cDNA]
RNA removal:
?. [Cleanup cDNA]
Add , pipette-mix.
[1 M NaOH]
?. [Cleanup cDNA]
Incubate at  for  on a PCR block.
70 °C
?. [Cleanup cDNA]
Add , pipette-mix.
[1 M HCl]
?. [Silane Cleanup cDNA]
Prepare beads:
?. [Silane Cleanup cDNA]
Magnetically separate  per sample, remove the supernatant.
[MyONE Silane beads]
?. [Silane Cleanup cDNA]
Wash 1× with  and resuspend beads in .
[RLT buffer]
[RLT buffer]
?. [Silane Cleanup cDNA]
Bind RNA:
?. [Silane Cleanup cDNA]
Add beads in  to sample, mix and add .
[RLT buffer]
[100% EtOH]
?. [Silane Cleanup cDNA]
Pipette mix, leave pipette tip in a tube, pipette mix twice, for .
?. [Silane Cleanup cDNA]
Wash beads:
?. [Silane Cleanup cDNA]
Magnetically separate, remove the supernatant.
?. [Silane Cleanup cDNA]
Add , pipette resuspend and move to new tube.
[80% EtOH]
?. [Silane Cleanup cDNA]
After , magnetically separate, remove the supernatant, and wash 2× with 80% EtOH ().
?. [Silane Cleanup cDNA]
Magnetically separate, remove residual liquid with fine tip → air-dry .
?. [Silane Cleanup cDNA]
Elute RNA:
?. [Silane Cleanup cDNA]
Resuspend in , let it sit for  (do NOT remove from beads).
[5 mM Tris–HCl pH 7.5]