N2102Ep anti-ORF2 and rIgG IPs
Date: 10/07/20
• RNAse free L1 extraction buffer, 500mM NaCl. 1:250 RNasin in EB, 1:1000 in wash; protease inhibitor 1x in both extraction and wash buffer
• Cell line: N2102Ep • 100mg scale with 20ul anti-ORF2 or rIgG control beads; • 16 samples total, 8 replicates per type of beads (4 elute in Trizol; 4 elute in 2%SDS/50mM Tris, pH8)
IP
1. Weigh out 16x 100mg powder; add extraction buffer with RNAse inhibitor (1:250) and protease inhibitors (1:100); 1:4 (w/v) of extraction buffer to powder;
2. Sonicate samples for 5x2 sec at 2 Amp; repeat once (energy output 100mg samples: ~20J)
3. Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
• Combine clarified lysate from all tubes • Save 20ul of Input for Western (“input”); use the rest to set up 16x 100mg IP (8 with anti-ORF2 beads and 8 with rabbit IgG beads)
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 20uL of bead slurry to each buffer-filled tube. Wash 3x with 1mL
5. Set up IP with 20ul of anti-ORF2 beads (made on 02/19/20) or rIgG beads (10/04/19) per 100mg reaction
6. IP @ 4°C for 30’
7. After IP, take 5ul of FT from each tube, pool FT from 8 anti-ORF2 tubes (“FT_anti-ORF2”) and pool FT from 8 rIgG tubes (“FT_rIgG”)
8. Wash beads 3x 1ml with RNAsin containing wash buffer
9. Switch beads to fresh tubes at 2nd wash step
10. During the final wash step,10% of the beads was taken from each tube; pool beads from 8 anti-ORF2 tubes together and pool beads from 8 rIgG tubes together
11. Elute combined beads in 15ul of 2% SDS/40mM Tris, pH8; 70C for 5’ with mixing; collect eluate for western (“E_anti-ORF2” and “E_rIgG”)
RNA and MS sample processing (90% beads of each 100mg IP)
After last wash, Take 4 anti-ORF2 tubes and 4 rIgG tubes for RNA extraction • The remaining 90% of the beads was eluted directly in TRIzol, and RNA was subsequently extracted from this fraction (~6 µl per sample). • Additionally, the organic phase of the phasemaker tubes was also stored at -80C. This can be used for future MS analysis. • All steps from here may be performed at RT if not mentioned otherwise • Vortex for 1min, place the tubes aside (can be on ice or RT, Trizol extract is stable at RT)
The other set of 4x anti-ORF2 and 4x rIgG will be used for MS • Elute the remaining 90% of beads in 25ul of 2% SDS/40mM Tris, pH8; 70C for 5’ with mixing • Collect elution and put the tubes in speed vac to dry the samples down • Put dry pellet in -20 freezer. Continue with S-trap protocol
RNA extraction using Direct-zol RNA MicroPrep Kit (Trizol elution)
1. Spin the Phasemaker tube at 16k RCF, 30 sec
2. Collect the elution from step 12 (“E”, pulse spin, place on magnet) and transfer to the Phasemaker tube
3. 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.
4. Mix by hand, vigorously for 15 sec
5. 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.
6. Spin 16k RCF, 4C (not important, Trizol extract is stable at RT), 5 min
7. 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 for later m/s analysis.
8. Add an equal volume of 100% EtOH (~160-180uL) to each sample and mix thoroughly, vortex and pulse spin
9. 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.
10. Add 400ul Direct-zol RNA PreWash to the column and centrifuge @ 16,000rcf for 30 seconds. Discard the FT. Use the vacuum-trap system.
11. Repeat step 22
12. 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. 13. To elute RNA, add 6ul of RNase-Free Water directly to the column matrix and centrifuge @ 16,000rcf for 30 seconds.
14. Stored RNA samples at -80C and send to NYU later
MS sample preparation using S-trap micro columns (SDS elution)
MS sample preparation using S-Trap Micro Ultra-High Recovery protocol
Take tubes with dry SDS elution pellets from the freezer; resupsend in 25ul of SDS/Urea buffer and follow S-trap protocol
Send samples to NL for MS analysis
Resuspension solvent: 94.9:5.0:0.1 water:MeOH:FA
Resuspension volume: 20 uL
Injection volume: 5 uL
MS method: uPU-60mingradTOP20-highload_Luciano_IP_gradient
Link to RAW files: https://rockefeller.box.com/s/xqkobayui7uqrfu6mip1v5by9tuydtmr
Western analysis
Determine total protein concentration of input by Bradford Assay (11ug/ul); load 25ug on gel (2.3ul); load same volume of FT on gel
Add 50mM DTT and 1x LDS (all final concentrations) to each sample
Heat samples @ 70C for 10’
Load 4-12% Bis-Tris gel
1) Marker
2) Input
3) FT_anti-ORF2
4) FT_rIgG
5) E_anti-ORF2 (8x 10mg combined)
6) E_rIgG (8x 10mg combined)
7) Marker
8) E_MT302_anti-ORF1 (50% of 50mg IP, used as positive control for ORF1p signal)
9) Marker (not shown)
Wet transfer, 70V for 2h
Cut the membrane in half, between 75 and 50KD, use the top for anti-ORF2 and bottom for anti-ORF1
Block the membranes in TBST/5% milk @ RT, 2hr
Upper panel: Rabbit anti-ORF2 Western (Clone 5-5, 1.03mg/ml); freshly diluted antibody
• Primary Ab: Rabbit anti-ORF2 antibody, 1:500, 4°C, overnight
• Secondary Ab: ECL anti-rabbit HRP 1:10,000, RT, 1hr
Lower panel: Anti-ORF1 Western (Abmart, 4H1, 2mg/ml); freshly diluted antibody
• Primary Ab: Mouse anti-ORF1 antibody, 1:5,000, 4°C, overnight
• Secondary Ab: ECL anti-mouse HRP 1:10,000, RT 1hr
20 second exposure, high sensitivity, auto tone
Top panel, 5 minute exposure, super sensitivity, auto tone
