TITLE: qPCR Quantify cDNA, PCR, 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]
?. [Prepare qPCR Master Mix; 9 μL Per Sample]
Prepare qPCR Master Mix; 9 μL Per Sample:  AB1qPCR 2× master mix5.0 μL2H2O3.6 μL3qPCR primer mix0.4 μL (10 μM each qPCR-grade D5x/D7x mix)
AB1qPCR 2× master mix5.0 μL2H2O3.6 μL3qPCR primer mix0.4 μL (10 μM each qPCR-grade D5x/D7x mix)
?. [Prepare qPCR Master Mix; 9 μL Per Sample]
Mix, dispense into a 384-well qPCR plate.
?. [Prepare qPCR Master Mix; 9 μL Per Sample]
Add  , seal, mix.
[1:10 diluted (in H2O) sample cDNA]
?. [Prepare qPCR Master Mix; 9 μL Per Sample]
Run qPCR, note Cq values.
?. [PCR Amplify cDNA]
AB1Prepare PCR on ice; 50 μL total per sample:PCR conditions (cycle # depending on library):22× PCR master mix: 25.0 μL98 °C for 30 s3H2O: 7.5 μL98 °C for 15 s → 68 °C for 30 s → 72 °C for 40 s (×6 cycles)420 μM forward primer (D50x): 2.5 μL98 °C for 15 s → 72 °C for 60 s (×[qPCR Cq minus 9] cycles)520 μM reverse primer (D70x): 2.5 μL72 °C 1 min6Sample cDNA: 12.5 μL4 °C hold7Total cycle # for final PCR: 3 cycles less than the qPCR Ct or Cq of the 1:10 diluted sample
AB1Prepare PCR on ice; 50 μL total per sample:PCR conditions (cycle # depending on library):22× PCR master mix: 25.0 μL98 °C for 30 s3H2O: 7.5 μL98 °C for 15 s → 68 °C for 30 s → 72 °C for 40 s (×6 cycles)420 μM forward primer (D50x): 2.5 μL98 °C for 15 s → 72 °C for 60 s (×[qPCR Cq minus 9] cycles)520 μM reverse primer (D70x): 2.5 μL72 °C 1 min6Sample cDNA: 12.5 μL4 °C hold7Total cycle # for final PCR: 3 cycles less than the qPCR Ct or Cq of the 1:10 diluted sample
?. [PCR Amplify cDNA]
Dispense into 8-well strips, add  +  and mix.
[CLIP sample]
[H2O]
?. [PCR Amplify cDNA]
Perform PCR as indicated above.
?. [SPRI Cleanup Library]
Add  suspension (do not separate) per 50 μL PCR reaction, mix, incubate at  for  (pipette mix 2–3× during incubation).
[AmpureXP beads]
0 Room temperature
?. [SPRI Cleanup Library]
Magnetically separate, wash beads 2× with 75% EtOH remove the supernatant → air-dry beads .
?. [SPRI Cleanup Library]
Resuspend in , let it sit for , magnetically separate for .
[ H2O]
?. [SPRI Cleanup Library]
Transfer  to new tubes.
18 µl