TITLE: Effective identification of RNA-binding proteins using RNA Immunoprecipitation
AUTHORS: Georgios I Laliotis, Philip N. Tsichlis

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?. [Chemicals Required]
For 100ml1.28M sucrose        43.21g
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							1M Tris-HCL 7.5        4ml
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							100mM MgCl2         20ml
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							100% Triton-X            4ml
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							2. R.I.P. buffer; 150mM KCl, 25mM Tris-HCL (pH=7.5), 5mM EDTA, 0.5mM DTT, 0.5% Noditet P-40For 250ml
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							150mM KCl                2.795g
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							1M Tris-HCL 7.5        6.25ml
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							50mM EDTA 8.0        2.5ml
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							1M DTT                       125ul
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							100% NP-40               1.25ml
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							1x Protease inhibitor (add fresh every time)
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							100U/ml RNAase inhibitor (add fresh every time)
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							Autoclave and keep chilled at 4.
?. [Cross linking and Lysis of The cells]
Crosslinking is suggested to make stable interactions
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							For cell lines
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							For each Ab to be tested, 1x100mm plates must be used.
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							1. Add 270ul of 37% Formaldehyde into 10ml culture media (P100)
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							2. Incubate at 37 for 15’
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							3. Add 1ml 1.25M Glycine and incubate 5’ at RT
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							4. Wash cells with ice-cold PBS x2
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							5. Add 1ml of PBS (200ul), Nuclear isolation Buffer (200ul) and water (600ul)
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							6. Scrap cells and collect in a new tube
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							7. Centrifuge in 14,000rpm for 2’ at 4 deg.
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							8. Remove supernatant
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							9. Add/resuspend with 1ml freshly made RIP Buffer (10ul protease inhibitor+100U/ml RNAase inhibitor)
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							10. Incubate on ice for 10’
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							11. Sonication : 15 sec on/45 sec off (x6 times for each sample)
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							12. Centrifuge at 14,000rpm for 15’ at 4 deg.
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							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)
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?. [Immunoprecipitation of Crosslinked Protein/RNA Complexes]
1. Precleaning : add 30ul of protein G bead for precleaning
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							2. Incubate for 1h at 4 deg. With rotation
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							3. Centrifuge at 3000rpm for 2' at 4 deg.
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							4. Transfer supernatant in a new tube
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							5. Add 1-5ug of antibody or IgG (negative control)
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							6. As positive control use anti-SNRNP70 Ab Millipore Cat. # CS203214
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							7. Incubate overnight at 4 deg. With rotation
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							8. Add 50ul Magnetic Beads and incubate with rotation 1-4 hours at 4 deg.
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							9. Spin beads at 3000rpm for 2’ at 4 deg.
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							10. Resuspend in 500ml RIP buffer. 
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							11. Rotate at 4 deg. For 5 minutes each. Repeat x3 for a final of 4 washes.
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							12. Resuspend beads with 100ul RIP buffer+1ul RNAase inhibitor (do not forget 10% input)
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							13. Incubate at 70 deg. For 1h to de-crosslink samples
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							14. Spin down and remove supernatant.
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?. [RNA purification and Reverse Transcription]
RNA Purification1. 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). 
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							2. Incubate at a heating block in 55oC for 30’ with shaking.
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							3. Repeat step 1-2  two times. For the input samples do the step once.
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							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)
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							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.
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							6. Centrifuge at 14,000rpm for 30 min at 4 deg. And discard the supernatant carefully.
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							7. Wash the pellet once with 80% ethanol. Centrifuge at 14,000rpm for 15 min at 4 deg.
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							8. Discard the supernatant carefully and air dry the pellets
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							9. Re-suspend in 20ul RNAase-free water and place tubes on ice.
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							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
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							Reverse Transcription1. 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.  AB1ReagentVolume/reaction (ul)2RNA9.035x RT Buffer4.0420x Enzyme mix1.05RT Primer Mix1.06RNAse free-water5.07Final Volume20.03. In a Thermo cycle set the following programRT Reaction 37 60min
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							Stop the reaction 95 5 min
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							Hold   4 Hold
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							4. Remove the PCR Tubes. Dilute the reaction with 180ul Nuclease free water (10x dilution). Reactions can be stored at -20.
AB1ReagentVolume/reaction (ul)2RNA9.035x RT Buffer4.0420x Enzyme mix1.05RT Primer Mix1.06RNAse free-water5.07Final Volume20.0
?. [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.
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							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.
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							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’
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							2. Add 2ul of appropriate sample to the PCR tube and return on ice.
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							3. Add the following reagents
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							  AB1ReagentVolume/reaction (ul)2DNA2.0310x PCR Buffer(-MgCl2)10.04MgCl2 (50mM)0.652.5mM dNTPs1.66RIP Primers U1 snRNA0.87Taq (5U/uL)0.44. Set up the following PCR reaction Initial Denaturation  94deg.  3’
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							 Denature 94 deg. 20’’     
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							 Anneal 60 deg.  30’’ x20
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							 Extension 72 deg.  30’’
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							 Final Extension 72 deg.  2’
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							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.
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AB1ReagentVolume/reaction (ul)2DNA2.0310x PCR Buffer(-MgCl2)10.04MgCl2 (50mM)0.652.5mM dNTPs1.66RIP Primers U1 snRNA0.87Taq (5U/uL)0.4
?. [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.
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							2. Prepare the following master mix.
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							  AB1ReagentVolume/reaction (ul)2cDNA23SYBR-Green Master Mix12.54RT Primer Mix1.05RNAse free-water9.56Final Volume23.03. Run on this programInitial Denaturation 95deg.  10’
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							Denature 95 deg.  15’’
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							Anneal and extension.        Tm-5 1’          x40
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AB1ReagentVolume/reaction (ul)2cDNA23SYBR-Green Master Mix12.54RT Primer Mix1.05RNAse free-water9.56Final Volume23.0