RNA extractions in endogenous cell lines following mIgG IP
Date: 09/10/20
• RNAse free L1 extraction buffer. 1:250 RNasin in EB, 1:1000 in wash.
• 100mg scale with 20ul mIgG beads (20x16=320ul total)
Samples
• 1-4. N2102Ep
• 5-8. NTERA2
• 9-12. PA-1 PC
• 13-16. PA-1 DC (200mg cell powder with matrigel; equivalent to 100mg cell powder alone)
All buffers made with RNAse-free H20.
METHODS
Precautions
• Before starting spray bench and pipettes subsequently with RNAseZAP, H2O and 70% EtOH. • Use nuclease free tubes and pipette tips
Mock IP with mIgG beads
1. Prep extraction buffer with RNAse inhibitor and protease inhibitors; Add 1:4 (w/v) of extraction buffer to powder after powder has EQ’d at RT for 30sec
2. Sonicate samples for 5x2 sec at 2 Amp; repeat once (energy output 100mg samples: ~20J; 200mg samples: ~40J;)
3. Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R);
4. During spin, wash 20ul of beads per sample with extraction buffer that does NOT have RNasin added (to conserve RNasin). (Beads stored in 1X PBS, 0.5ng/uL BSA, 50% glycerol.) To wash beads, add 1mL wash buffer to each tube first, then add 10uL of bead slurry to each buffer-filled tube. Wash 3x with 1mL
5. After spin, save 10ul of supernatant (“Prot Input + #”) for protein analysis, and 35ul of supernatant for RNA prep; add equal volume of TRIzol to the RNA portion (RNA Input + #) and snap freeze in LN2.
6. Set up IP with 20ul of anti-ORF1 beads/sample (made on 12/11/19)
7. IP@ 4°C for 30’ (get tubes and Zymo columns ready while IP is going)
8. Save 10ul of flow-through (“Prot FT + #”) for protein analysis; RNA portion of FT is not needed
9. Wash beads 3x 1ml with RNAsin containing wash buffer
10. Switch beads to fresh tubes at 2nd wash step
11. Trizol Elution: Elute in 250ul of Trizol (“E + #”)
All steps from here may be performed at RT if not mentioned otherwise
12. Vortex for 1min, place the tubes aside (can be on ice or RT, Trizol extract is stable at RT)
RNA extraction
13. Spin the Phasemaker tube at 16k RCF, 30 sec
14. Collect the elution from step 12 (“E”, pulse spin, place on magnet) and transfer to the Phasemaker tube
15. Add 25ul water and then 50ul Chloroform to the Phasemaker. Do this immediately after adding TriZol elutions. Not adding the water will cause the PhaseMaker to fail and 'swallow' the aqueous portion of the TriZol reagent mix.
16. Mix by hand, vigorously for 15 sec
17. Incubate 2 min @ RT with end-over-end mixing This step is almost certainly dispensable for IP - it is supposed to be time for RNPs to dissociate.
18. Spin 16k RCF, 4°C (not important, Trizol extract is stable at RT), 5 min
19. Transfer the aqueous phase to a new tube by pressing pipette tip against wall of tube; avoid puncturing the Phasemaker gel. Store numbered phasemaker tubes with remaining organic phase at -20°C for later m/s analysis.
20. Add an equal volume of 100% EtOH (~160-180uL) to each sample and mix thoroughly, vortex and pulse spin
21. Transfer the mixture to a Zymo-Spin IC column in a collection tube and centrifuge @ 16,000rcf for 30 seconds (max volume to load on column is 700ul). Discard the FT. Use the vacuum-trap system.
22. Add 400ul Direct-zol RNA PreWash to the column and centrifuge @ 16,000rcf for 30 seconds. Discard the FT. Use the vacuum-trap system.
23. Repeat step 22
24. Add 700ul RNA Wash Buffer to the column and centrifuge for 2 minutes @ 16,000rcf to ensure complete removal of the wash buffer. Transfer the column carefully into an RNase-free tube.
25. To elute RNA, add 6ul of RNase-Free Water directly to the column matrix and centrifuge @ 16,000rcf for 30 seconds.
26. Stored RNA samples at -80°C
27. Send all 6ul (RNA + #) to NYU for bioanalyzer and for RNA-seq analyses
We will send 3 sets of 16 samples (48 total) to NYU; 6ul each
Two sets of samples from this experiment (control IP with mIgG beads):
• RNA samples from Trizol elution after IP with mouse IgG beads: Labeled 1-16; with cell line name and “mIgG”
• Total RNA samples from cell extract Label 1-16 (in blue marker); with cell line name and prep date “9/11”
One set of samples from previous experiment (anti-ORF1 IP in sample cell lines):
• Total RNA samples from cell extract Label 1-16 (in black marker); with cell line name and prep date “8/31”
Sample #15 in this set could be bad. The tube holding cell extract in Trizol cracked into pieces before RNA prep. I recovered the Trizol with sample from the well of the centrifuge rotator and proceed since I need a balance tube anyway.
Since we did another set of total RNA prep this time, losing one replicate from the previous experiment may not be crucial.
AmAc/MeOH precipitation of protein from mIgG elution in organic phase
Date: 09/14/20
1. Take Phasemaker tubes with organic phase out from -80°C freezer
2. Use a gel loading tip, pass it through phasemaker to reach the bottom layer (organic phase); check the volume
3. Add 200ul of cold 0.1M AmAc in MeOH (between 1 and 1.5 volume of the organic phase)
4. Mix by hand, vigorously for 15 sec
5. Spin 16k RCF, 4°C, 5 min
6. The organic phase moved to the top.
7. Transfer the top layer to a fresh tube; add 1.5ul of glycoblue to each sample
8. Add 5 volume of precipitation solution (final); mix by inverting, and the sample is incubated overnight at –20°C. Calculated the volume of precipitation solution to add (subtracting 200ul already added)
• 5 volume final: 5 x165 -200 = 625 ul
9. Precipitate was pelleted, 20k x g for 30' @ 4°C
10. Make the side of Eppendorf tube that the pellet should be at; carefully remove the supernatant (Saw small blue pellet in each tube)
11. After centrifugation, wash pellet with1ml of cold 0.1M AmAc/MeOH; 20k rcf for 15' @ 4°C
12. Wash pellet again with 1ml of cold Acetone; 20k rcf for 15' @ 4°C
13. Discard supernatant, the pellet is dried under vacuum.
14. Resuspend pellet with 25ul of SDS/Urea/Glycine buffer followed by S-trap protocol.
S-Trap Micro Ultra-High Recovery Protocol
MS sample preparation using S-Trap Micro Ultra-High Recovery protocol
Date: 09/17/20
1. Add 25 μL of fresh urea-SDS lysis/solublization buffer: 5% SDS, 8 M urea, 100 mM glycine* pH 7.55. Vortex to resuspend the pellet
2. Reduce and alkylate disulfides. Avoid high temperatures are not recommended due to urea. Add DTT to 25mM final concentration
Incubate @ 37°C for 30 minutes (70 °C is too hot for this application)
Put the tube on ice briefly to bring the temperature down
Add Iodoacetamide to 100mM final concentration
Incubate @ RT in dark for 30 minutes.
atight seal. 10. Elute peptides with 40 μL each of 50 mM TEAB and then 0.2% aqueous formic acid (FA). Add the first TEAB elution to the trypsin solution prior to any centrifugation. Centrifuge elutions through at 4,000 g.
11. Elute hydrophobic peptides with 35 μL 50% acetonitrile (ACN), 0.2% formic acid.
12. Dry down peptides and resuspend as desired (buffer A or MALDI matrix).
Total volume before drying down about 140ul:
25ul digestion
40ul E1 (50 mM TEAB, pH8)
40ul E2 (0.2% FA)
35ul E3 (50% ACN, 0.2% FA)
Samples were sent to NL for MS
Resuspension solvent: 94.9:5.0:0.1 water:MeOH:FA
Resuspension volume: 20 uL
Injection volume: 1, 2 or 5 uL as indicated in the Raw file’s name
MS method: uPU-60mingradTOP20-highload_Luciano_IP_gradient
Link to RAW files: https://rockefeller.box.com/s/xqkobayui7uqrfu6mip1v5by9tuydtmr