Jun 15, 2022
Version 2
Biotin-Labelling of Immunoprecipitated RNA (v1pre) V.2
This protocol is a draft, published without a DOI.
- Steven Blue1,
- Eric Van Nostrand2,
- Gene Yeo1
- 1University of California San Diego;
- 2Baylor College of Medicine
Protocol Citation: Steven Blue, Eric Van Nostrand, Gene Yeo 2022. Biotin-Labelling of Immunoprecipitated RNA (v1pre). protocols.io https://protocols.io/view/biotin-labelling-of-immunoprecipitated-rna-v1pre-cbehsjb6Version created by Steven Blue
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: June 15, 2022
Last Modified: June 15, 2022
Protocol Integer ID: 64681
Keywords: CLIP, RNA visualization, RNP visualization, biotin labeling
Abstract
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
Materials
MATERIALS
RNase Inhibitor, Murine - 15,000 unitsNew England BiolabsCatalog #M0314L
T4 RNA Ligase 1 (ssRNA Ligase) (30,000 units/ml) - 5,000 unitsCatalog #M0437M
T4 Polynucleotide Kinase - 2,500 unitsNew England BiolabsCatalog #M0201L
Dynabeads™ M-280 Sheep Anti-Mouse IgGThermo FisherCatalog #11201D
Dynabeads™ M-280 Sheep Anti-Rabbit IgGThermo FisherCatalog #11204D
Chemiluminescent Nucleic Acid Detection Module KitThermo FisherCatalog #89880
TURBO™ DNase (2 U/µL)Thermo FisherCatalog #AM2239
Ambion™ RNase I, cloned, 100 U/µLThermo FisherCatalog #AM2295
FastAP Thermosensitive Alkaline Phosphatase (1 U/µL)Thermo FisherCatalog #EF0652
NuPAGE™ MOPS SDS Running Buffer (20X)Thermo FisherCatalog #NP000102
NuPAGE™ Transfer Buffer (20X)Thermo FisherCatalog #NP00061
NuPAGE™ LDS Sample Buffer (4X)Thermo FisherCatalog #NP0008
NuPAGE™ 4-12% Bis-Tris Protein Gels, 1.0 mm, 12-wellThermo FisherCatalog #NP0322BOX
pCp-BiotinJena BioscienceCatalog #NU-1706-BIO
- Lysis buffer:50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40 (Igepal CA630), 0.1% SDS, 0.5% sodium deoxycholate (protect from light), (add fresh) 1:200 Protease Inhibitor Cocktail III, in RNase/DNase free H2O.
- High salt wash buffer:50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate (protect from light), in RNase/DNase free H2O.
- Wash buffer:20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.2% Tween-20, in RNase/DNase free H2O.
- Protease Inhibitor Cocktail III EMD Millipore 539134
- 1M DTT
Conjugate Antibody to Beads
Conjugate Antibody to Beads
For each 4M cell lysate, aliquot 25 μL of species-specific Dynabeads (e.g. ThermoFisher catalog # 11203D, 11202D, or equivalent)
Wash the beads twice in 500 μL cold Lysis Buffer.
Resuspend in 100 μL of Lysis Buffer.
Add 2 μg of RBP-specific antibody to each tube.
Rotate at room temperature for 45 min. Proceed to step 7 (Cell Lysis) while rotating.
Wash twice with 500 μL cold Lysis Buffer and reserve on ice until step 14.
Cell Lysis
Cell Lysis
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
Lyse on ice for 15 min.
Sonicate on Low setting for 5 min, 30 sec on/30 sec off.
Make 2 dilutions of RNase I in PBS:
- 1:25 (4 U/μL)
- 1:3 (33.3 U/μL)
Add 1 μL Turbo DNase to each 4M cell lysate.
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
Immediately incubate lysates in a Thermomixer set to 37°C, shaking at 1200 rpm, for 5 min.
Centrifuge at 15,000 x g for 10 min at 4°C. Transfer cleared lysate to washed beads.
Rotate at 4°C overnight.
Post-IP Washes
Post-IP Washes
For each sample, wash twice with 500 μL cold High Salt Wash Buffer.
To each sample, add 500 μL cold High Salt Wash Buffer, mix, add 500 μL cold Wash Buffer.
For each sample, wash 3X with cold Wash Buffer, keep on ice after last wash.
Dephosphorylation of IP Samples
Dephosphorylation of IP Samples
Briefly spin tubes and remove residual wash buffer. Resuspend the beads in the following mix, volumes per sample:
| H2O | 38.0 μL | |
| 10X FastAP Buffer | 5.0 μL | |
| Murine RNase Inhibitor | 2.0 μL | |
| Turbo DNase | 2.0 μL | |
| FastAP Enzyme | 3.0 μL |
Incubate the FastAP reaction in a Thermomixer at 37°C, mixing at 1200 rpm, for 10 min.
While incubating the FastAP reaction, prepare the following mix, volumes per sample:
| H2O | 116.0 μL | |
| 5X PNK pH 6.5 Buffer | 30.0 μL | |
| T4 PNK Enzyme | 4.0 μL |
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.
Post-PNK Washes
Post-PNK Washes
To each sample, add 200 μL High Salt Wash Buffer, mix, separate on magnet, and remove supernatant.
Add 500 μL High Salt Wash Buffer, mix, then add 500 μL Wash Buffer. Remove supernatant.
Wash 3X with 500 μL Wash Buffer.
Biotinylated Cytidine Ligation
Biotinylated Cytidine Ligation
In an RNase-free 1.5 mL microcentrifuge tube, mix the following mix, volumes per sample:
| H2O | 9.6 μL | |
| 10X RNA Ligase Buffer (no DTT) | 3.0 μL | |
| 0.1 M ATP | 0.3 μL | |
| 100% DMSO | 0.9 μL | |
| 1% Tween-20 | 0.6 μL | |
| 50% PEG 8000 | 9.0 μL | |
| Murine RNase Inhibitor | 0.4 μL | |
| RNA Ligase High Conc. Enzyme | 2.4 μL | |
| Biotinylated Cytidine (Bis)phosphate | 0.5 μL |
Magnetically separate each IP sample, remove Wash Buffer, and resuspend the beads in 26 μL of master mix.
Incubate samples at 16°C with gentle shaking for 2 hrs or overnight (recommended).
Post-Ligation Cleanup
Post-Ligation Cleanup
Add 500 μL cold High Salt Wash Buffer, mix, magnetically separate, and remove supernatant.
Add 500 μL cold High Salt Wash Buffer, move on magnet, add 500 μL cold Wash Buffer, magnetically separate, remove supernatant.
Wash 3X with 500 μL cold Wash Buffer.
Resuspend in 20 μL Wash Buffer.
Gel Electrophoresis & Transfer
Gel Electrophoresis & Transfer
Add 10.5 μL of denaturing mix for SDS-PAGE (7.5 μL 4X LDS buffer, 3 μL 1M DTT)
Incubate at 70°C, mixing at 1200 rpm for 10 min.
Place tubes on ice for > 1 min.
Magnetically separate samples and load 15 μL on gel, reserving the other half at -20°C as backup.
Run the gel at 150V for 75 min.
Transfer to nitrocellulose membrane at 30V overnight.
Membrane Development
Membrane Development
Develop membrane as follows using the Chemiluminescent Nucleic Acid Detection Module kit (cat. no. 89880)
Slowly warm the Blocking Buffer and the 4X Wash Buffer to 37-50°C in a water bath until all particulates are dissolved.
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).
Prepare conjugate/blocking buffer solution by adding 31.25 μL of the Stabilized Streptavidin-Horseradish Peroxidase Conjugate to 10mL Blocking Buffer.
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.
Prepare 1X wash solution by adding 40 mL of 4X Wash Buffer to 120 mL water.
Transfer membrane to a new container and rinse briefly with 20 mL of 1X wash solution.
Wash membrane four times for 5 min each in 20 mL of 1X wash solution with gentle shaking.
Transfer membrane to a new container and add 30 mL of Substrate Equilibration Buffer. Incubate membrane for 5 min with gentle shaking.
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.
Remove membrane from the Substrate Equilibration Buffer and remove excess buffer. Place membrane in a clean container or clean sheet of plastic wrap.
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.
Remove membrane from the Working Solution and remove excess buffer. Do not allow the membrane to dry out.
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.