TITLE: Biotin-Labelling of Immunoprecipitated RNA (v1pre)
AUTHORS: Steven  Blue, Eric  Van Nostrand, Gene Yeo

[DESCRIPTION]
RNA binding proteins (RBPs) regulate a diverse array of RNA processing steps by binding RNAs through sequence and structural elements. The development of crosslinking and immunoprecipitation (CLIP) enabled identification of RBP targets transcriptome-wide in a robust manner. During CLIP, it is often desired to visualize RBP:RNA complexes after denaturing SDS-PAGE electrophoresis and transfer to nitrocellulose membranes. However, standard methods used radiolabeling of RNA followed by autoradiography, introducing significant challenges for usability and scaling experiments to profile many RBPs. Here we describe an alternative approach to visualize RBP:RNA complexes using ligation of biotinylated nucleotides, followed by standard chemiluminescent imaging. This approach retains the advantages of visualization while decreasing handling complexity, enabling large-scale experiments to verify the presence and sizing of immunoprecipitated RBP:RNA complexes.

[GUIDELINES]
For best results, include the following experimental conditions: 
UV-crosslinked cell lysate (4M cells worth), treated with 8U RNase I
UV-crosslinked cell lysate (4M cells worth), treated with 66.6U RNase I 
Non-crosslinked cell lysate (4M cells worth), treated with 8U RNase I

[STEPS]
SECTION: Conjugate Antibody to Beads
1. For each 4M cell lysate, aliquot 25 μL of species-specific Dynabeads (e.g. ThermoFisher catalog # 11203D, 11202D, or equivalent)
SECTION: Conjugate Antibody to Beads
2. Wash the beads twice in 500 μL cold Lysis Buffer.
SECTION: Conjugate Antibody to Beads
3. Resuspend in 100 μL of Lysis Buffer.
SECTION: Conjugate Antibody to Beads
4. Add 2 μg of RBP-specific antibody to each tube.
SECTION: Conjugate Antibody to Beads
5. Rotate at room temperature for 45 min. Proceed to step 7 (Cell Lysis) while rotating. 
SECTION: Conjugate Antibody to Beads
6. Wash twice with 500 μL cold Lysis Buffer and reserve on ice until step 14.
SECTION: Cell Lysis
7. Lyse cells in cold eCLIP lysis buffer containing 1X protease inhibitor.
5.5 μL protease inhibitor cocktail III per 1 mL Lysis Buffer. 
Lyse pellets at similar ratio to that used in CLIP experiments (200 μL Lysis Buffer per 4M cells)
A typical experiment includes 2 (4 million cell) crosslinked samples and 1 (4 million cell) non-crosslinked sample
SECTION: Cell Lysis
8. Lyse on ice for 15 min.
SECTION: Cell Lysis
9. Sonicate on Low setting for 5 min, 30 sec on/30 sec off.
SECTION: Cell Lysis
10. Make 2 dilutions of RNase I in PBS:
1:25 (4 U/μL)
1:3 (33.3 U/μL)
SECTION: Cell Lysis
11. Add 1 μL Turbo DNase to each 4M cell lysate.
SECTION: Cell Lysis
12. Add diluted RNase to lysate:

2 μL of 1:25 dilution to the 1st crosslinked lysate = Crosslink Standard RNase
2 μL of 1:3 dilution to the 2nd crosslinked lysate = Crosslink High RNase
2 μL of 1:25 dilution to the non-crosslinked lysate = Non-crosslinked Standard RNase

SECTION: Cell Lysis
13. Immediately incubate lysates in a Thermomixer set to 37°C, shaking at 1200 rpm, for 5 min.
SECTION: Cell Lysis
14. Centrifuge at 15,000 x g for 10 min at 4°C. Transfer cleared lysate to washed beads. 
SECTION: Cell Lysis
15. Rotate at 4°C overnight.
SECTION: Post-IP Washes
16. For each sample, wash twice with 500 μL cold High Salt Wash Buffer.
SECTION: Post-IP Washes
17. To each sample, add 500 μL cold High Salt Wash Buffer, mix, add 500 μL cold Wash Buffer.
SECTION: Post-IP Washes
18. For each sample, wash 3X with cold Wash Buffer, keep on ice after last wash.
SECTION: Dephosphorylation of IP Samples
19. Briefly spin tubes and remove residual wash buffer. Resuspend the beads in the following mix, volumes per sample:

 

SECTION: Dephosphorylation of IP Samples
20. Incubate the FastAP reaction in a Thermomixer at 37°C, mixing at 1200 rpm, for 10 min.
SECTION: Dephosphorylation of IP Samples
21. While incubating the FastAP reaction, prepare the following mix, volumes per sample:

 

SECTION: Dephosphorylation of IP Samples
22. Without removing the FastAP mix, add 150 μL of the PNK mix to each sample and place back on Thermomixer, incubating at 37°C, 1200 rpm, for 20 min. 
SECTION: Post-PNK Washes
23. To each sample, add 200 μL High Salt Wash Buffer, mix, separate on magnet, and remove supernatant. 
SECTION: Post-PNK Washes
24. Add 500 μL High Salt Wash Buffer, mix, then add 500 μL Wash Buffer. Remove supernatant.
SECTION: Post-PNK Washes
25. Wash 3X with 500 μL Wash Buffer.
SECTION: Biotinylated Cytidine Ligation
26. In an RNase-free 1.5 mL microcentrifuge tube, mix the following mix, volumes per sample:


 


SECTION: Biotinylated Cytidine Ligation
27. Magnetically separate each IP sample, remove Wash Buffer, and resuspend the beads in 26 μL of master mix. 
SECTION: Biotinylated Cytidine Ligation
28. Incubate samples at 16°C with gentle shaking for 2 hrs or overnight (recommended).
SECTION: Post-Ligation Cleanup
29. Add 500 μL cold High Salt Wash Buffer, mix, magnetically separate, and remove supernatant.
SECTION: Post-Ligation Cleanup
30. Add 500 μL cold High Salt Wash Buffer, move on magnet, add 500 μL cold Wash Buffer, magnetically separate, remove supernatant.
SECTION: Post-Ligation Cleanup
31. Wash 3X with 500 μL cold Wash Buffer.
SECTION: Post-Ligation Cleanup
32. Resuspend in 20 μL Wash Buffer. 
SECTION: Gel Electrophoresis &amp; Transfer
34. Incubate at 70°C, mixing at 1200 rpm for 10 min.
SECTION: Gel Electrophoresis &amp; Transfer
33. Add 10.5 μL of denaturing mix for SDS-PAGE (7.5 μL 4X LDS buffer, 3 μL 1M DTT)
SECTION: Gel Electrophoresis &amp; Transfer
35. Place tubes on ice for > 1 min.
SECTION: Gel Electrophoresis &amp; Transfer
36. Magnetically separate samples and load 15 μL on gel, reserving the other half at -20°C as backup.
SECTION: Gel Electrophoresis &amp; Transfer
37. Run the gel at 150V for 75 min. 
SECTION: Gel Electrophoresis &amp; Transfer
38. Transfer to nitrocellulose membrane at 30V overnight.
SECTION: Membrane Development
39. Develop membrane as follows using the Chemiluminescent Nucleic Acid Detection Module kit (cat. no. 89880)
SECTION: Membrane Development
39.1. Slowly warm the Blocking Buffer and the 4X Wash Buffer to 37-50°C in a water bath until all particulates are dissolved. 
SECTION: Membrane Development
39.2. Block membrane by adding 10 mL Blocking Buffer and incubate for 15 min with gentle shaking at room temperature (all further steps done at room temperature).
SECTION: Membrane Development
39.3. Prepare conjugate/blocking buffer solution by adding 31.25 μL of the Stabilized Streptavidin-Horseradish Peroxidase Conjugate to 10mL Blocking Buffer.
SECTION: Membrane Development
39.4. Decant blocking buffer from the membrane and add 10 mL to the conjugate/blocking solution. Incubate membrane in the conjugate/blocking buffer solution for 15 min with gentle shaking.
SECTION: Membrane Development
39.5. Prepare 1X wash solution by adding 40 mL of 4X Wash Buffer to 120 mL water.
SECTION: Membrane Development
39.6. Transfer membrane to a new container and rinse briefly with 20 mL of 1X wash solution.
SECTION: Membrane Development
39.7. Wash membrane four times for 5 min each in 20 mL of 1X wash solution with gentle shaking.
SECTION: Membrane Development
39.8. Transfer membrane to a new container and add 30 mL of Substrate Equilibration Buffer. Incubate membrane for 5 min with gentle shaking.
SECTION: Membrane Development
39.9. Prepare Chemiluminescent Substrate Working Solution by adding 2 mL Luminol/Enhancer Solution to 6 mL Stable Peroxide Solution. Note: Working solution is susceptible damage via prolonged light exposure. Keep solution in an amber bottle or keep away from light.
SECTION: Membrane Development
39.10. Remove membrane from the Substrate Equilibration Buffer and remove excess buffer. Place membrane in a clean container or clean sheet of plastic wrap.
SECTION: Membrane Development
39.11. Pour the Substrate Working Solution onto the membrane so that it completely covers the surface. Incubate membrane in the substrate solution for 5 min without shaking. 
SECTION: Membrane Development
39.12. Remove membrane from the Working Solution and remove excess buffer. Do not allow the membrane to dry out.
SECTION: Membrane Development
39.13. Wrap the membrane in plastic wrap, avoiding bubbles, and place in a film cassette. Obtain optimal signal by adjusting film exposure time or by exposing membrane to multiple films simultaneously.