Aug 13, 2020

Public workspaceEffective identification of RNA-binding proteins using RNA Immunoprecipitation

DOI
dx.doi.org/10.17504/protocols.io.bjpbkmin
  • Georgios I Laliotis1,
  • Philip N. Tsichlis1
  • 1The Ohio State University
  • Georgios I Laliotis: Corresponding Author
George Laliotis
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DOI: dx.doi.org/10.17504/protocols.io.bjpbkmin
Protocol Citation: Georgios I Laliotis, Philip N. Tsichlis 2020. Effective identification of RNA-binding proteins using RNA Immunoprecipitation. protocols.io https://dx.doi.org/10.17504/protocols.io.bjpbkmin
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 have used this in our recent publication : ' Laliotis, Georgios I., et al. "IWS1 phosphorylation promotes cell proliferation and predicts poor prognosis in EGFR mutant lung adenocarcinoma patients, through the cell cycle-regulated U2AF2 RNA splicing." bioRxiv (2020).'
Created: August 13, 2020
Last Modified: August 13, 2020
Protocol Integer ID: 40387
Chemicals Required
Chemicals Required
1d
1d
  1. Nuclear isolation buffer; 1.28M Sucrose, 40mM Tris-HCL (pH=7.5), 20mM MgCl2, 4% Triton-X

For 100ml

1.28M sucrose 43.21g
1M Tris-HCL 7.5 4ml
100mM MgCl2 20ml
100% Triton-X 4ml

2. R.I.P. buffer; 150mM KCl, 25mM Tris-HCL (pH=7.5), 5mM EDTA, 0.5mM DTT, 0.5% Noditet P-40

For 250ml
150mM KCl 2.795g
1M Tris-HCL 7.5 6.25ml
50mM EDTA 8.0 2.5ml
1M DTT 125ul
100% NP-40 1.25ml
1x Protease inhibitor (add fresh every time)
100U/ml RNAase inhibitor (add fresh every time)


Autoclave and keep chilled at 4.
Cross linking and Lysis of The cells
Cross linking and Lysis of The cells
6h
6h
Crosslinking is suggested to make stable interactions

For cell lines

For each Ab to be tested, 1x100mm plates must be used.

1. Add 270ul of 37% Formaldehyde into 10ml culture media (P100)
2. Incubate at 37 for 15’
3. Add 1ml 1.25M Glycine and incubate 5’ at RT
4. Wash cells with ice-cold PBS x2
5. Add 1ml of PBS (200ul), Nuclear isolation Buffer (200ul) and water (600ul)
6. Scrap cells and collect in a new tube
7. Centrifuge in 14,000rpm for 2’ at 4 deg.
8. Remove supernatant
9. Add/resuspend with 1ml freshly made RIP Buffer (10ul protease inhibitor+100U/ml RNAase inhibitor)
10. Incubate on ice for 10’
11. Sonication : 15 sec on/45 sec off (x6 times for each sample)
12. Centrifuge at 14,000rpm for 15’ at 4 deg.
13. Transfer supernatant into a new single tube (keep 10% input and 10ul for Western blot analyses of the RNA-Binding protein of interest-add 10ul 2x Loading buffer+DTT.Boil for 10’ in 99 and perform Western Analysis)


Immunoprecipitation of Crosslinked Protein/RNA Complexes
Immunoprecipitation of Crosslinked Protein/RNA Complexes
1d 6h
1d 6h

1. Precleaning : add 30ul of protein G bead for precleaning
2. Incubate for 1h at 4 deg. With rotation
3. Centrifuge at 3000rpm for 2' at 4 deg.
4. Transfer supernatant in a new tube
5. Add 1-5ug of antibody or IgG (negative control)
6. As positive control use anti-SNRNP70 Ab Millipore Cat. # CS203214
7. Incubate overnight at 4 deg. With rotation
8. Add 50ul Magnetic Beads and incubate with rotation 1-4 hours at 4 deg.
9. Spin beads at 3000rpm for 2’ at 4 deg.
10. Resuspend in 500ml RIP buffer. 
11. Rotate at 4 deg. For 5 minutes each. Repeat x3 for a final of 4 washes.
12. Resuspend beads with 100ul RIP buffer+1ul RNAase inhibitor (do not forget 10% input)
13. Incubate at 70 deg. For 1h to de-crosslink samples
14. Spin down and remove supernatant.
RNA purification and Reverse Transcription
RNA purification and Reverse Transcription
4h
4h

RNA Purification

1. Resuspend beads in 100ul RIP buffer with 0.1% SDS and 30ug protease K. (For 1ml add 10ul from 10% SDS and 3ul from 10mg/ml protease K). 
2. Incubate at a heating block in 55oC for 30’ with shaking.
3. Repeat step 1-2  two times. For the input samples do the step once.
4. Add one volume of phenol-chloroform-isoamyl alcohol mixture and vortex to mix. Centrifuge for 1 min to separate phases. Recover (upper) water phase (you can aspirate with no problem 175ul)
5. 10ul yeast tRNA (1mg/ml), 12ul 3M sodium acetate and 250ul ethanol to 100ul water phase and mix. Ethanol-precipitate at -80 overnight. Note : other carriers such as linear acrylamide or glycogen (5ul from 20ug/ul stock-I use Invitoegen Glycogen) can be used instead of yeast tRNA.
6. Centrifuge at 14,000rpm for 30 min at 4 deg. And discard the supernatant carefully.
7. Wash the pellet once with 80% ethanol. Centrifuge at 14,000rpm for 15 min at 4 deg.
8. Discard the supernatant carefully and air dry the pellets
9. Re-suspend in 20ul RNAase-free water and place tubes on ice.
10. Typically, proceed to RNA quality control of the input with Nanodrop. Ideally we expect then 260/230 Ratio to be close to 2.00

Reverse Transcription

1. Label the appropriate number of PCR tubes 0.2ml for the number of samples to be Analysed and place on ice.

2. Set the reaction below.
ReagentVolume/reaction (ul)
RNA9.0
5x RT Buffer4.0
20x Enzyme mix1.0
RT Primer Mix1.0
RNAse free-water5.0
Final Volume20.0



3. In a Thermo cycle set the following program



RT Reaction 37 60min
Stop the reaction 95 5 min
Hold   4 Hold


4. Remove the PCR Tubes. Dilute the reaction with 180ul Nuclease free water (10x dilution). Reactions can be stored at -20.
Positive Control RT-PCR
Positive Control RT-PCR

1. Label the appropriate number of 0.2ml PCR tubes for the number of samples to be analyzed and place on ice.
At a minimum, there will be 4 samples to undergo PCR using the RIP Primers : cDNA from positive (anti-SNRNP70) and negative control antibody (Normal Rabbit IgG) immunoprecipitations, input and a no template tube as a Control fro DNA contamination.
The RIP primers are specific for the human U1 snRNP gene. F : 5’-GGG AGA TAC CAT GAT CAC GAA GGT-3’, R : 5’-CCA CAA ATT ATG CAG TCG AGT TTC CC-3’
2. Add 2ul of appropriate sample to the PCR tube and return on ice.
3. Add the following reagents
ReagentVolume/reaction (ul)
DNA2.0
10x PCR Buffer(-MgCl2)10.0
MgCl2 (50mM)0.6
2.5mM dNTPs1.6
RIP Primers U1 snRNA0.8
Taq (5U/uL)0.4

4. Set up the following PCR reaction

 Initial Denaturation  94deg.  3’
 Denature 94 deg. 20’’     
 Anneal 60 deg.  30’’ x20
 Extension 72 deg.  30’’
 Final Extension 72 deg.  2’


5. Run on 2% agarose gel. Τhe expected size of the PCR Product is 100 base pairs. You should see bands in anti-SNRNP70 RIP and in 10% input.
RIP-qPCR/RIP-Seq
RIP-qPCR/RIP-Seq
1. Add 2ul of the cDNA sample to the PCR plate suitable for the real time instrument of choice, performing in triplicates.
2. Prepare the following master mix.

ReagentVolume/reaction (ul)
cDNA2
SYBR-Green Master Mix12.5
RT Primer Mix1.0
RNAse free-water9.5
Final Volume23.0


3. Run on this program

Initial Denaturation 95deg.  10’
Denature 95 deg.  15’’
Anneal and extension.        Tm-5 1’          x40