INTRO
We want to check which ribosomal subunit ORF1p preferentially binds to. To check this, we do an anti-ORF1p IP, extract RNA and check RNA on the bioanalyzer, where we either fortify of disassemble the ribosomes. Supplement your lysis/wash buffer either with 25 mM EDTA to disassemble, or 10 mM MgCl2 to stabilize the ribosomes.
Extraction buffer: 20mM HEPES pH 7.4, 500mM NaCl, 1% Triton-X100, protease inhibitor, 1:250 RNasin, (+ 25mM EDTA or 10mM MgCl2).
Wash buffer: 20mM HEPES pH 7.4, 500mM NaCl, 1% Triton-X100, protease inhibitor, 1:1000 RNasin, (+ 25mM EDTA or 10mM MgCl2).
Samples (Depending on the availability of the MT302 powder these could be changed to duplicates. However, if the bioanalyzer is run at the genomics facility, they charge per chip (11 samples) so we might as well do triplicates).
1. MT302 #1 regular buffer/no EDTA or MgCl
2. MT302 #2 regular buffer/no EDTA or MgCl
3. MT302 #3 regular buffer/no EDTA or MgCl
4. MT302 EDTA #1
5. MT302 EDTA #2
6. MT302 EDTA #3
7. MT302 MgCl #1
8. MT302 MgCl #2
9. MT302 MgCl #3
all 50mg, 200ul EB, 4x1 seq 2Amp sonication
Everything RNA grade; clean bench, pipettes, etc with 70% EtOH-RNAse ZAP-H2O. Use filter tips and nuclease free H20.
Anti-ORF1 IP
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 4x1 sec at 2 Amp (energy output ~10J)
3. Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R); no need to save input, all of the sample will be used for RNA extraction.
4. During spin, wash 10ul of beads per sample (9x 10ul total) with extraction buffer that does NOT have RNasin added (to conserve RNasin). (Beads store d 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. Set up IP with 10ul of anti-ORF1 beads/sample (made on 2/10/20 )
6. IP@ 4°C for 30’ (get tubes and Zymo columns ready while IP is going)
7. Wash 3x 1ml with RNAsin containing wash buffer
8. Switch beads to fresh tubes at 2nd wash step
9. Trizol Elution: Elute in 250ul of Trizol All steps from here may be performed at RT if not mentioned otherwise
10. Vortex for 1min, place the tubes aside (can be on ice or RT, Trizol extract is stable at RT)
RNA extraction
11. Spin the Phasemaker tube at 16k RCF, 30 sec
12. Add 25ul nuclease free water and then 50ul Chloroform to the Phasemaker. Do this immediately after transfering elutions to the Phasemaker tube. Not adding the water will cause the PhaseMaker to fail and 'swallow' the aqueous portion of the TriZol reagent mix.
13. Collect the elution from step 9 (“E”, pulse spin, place on magnet) and transfer to the Phasemaker tube
14. Mix by hand, vigorously for 15 sec
15. 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.
16. Spin 16k RCF, 4C (not important, Trizol extract is stable at RT), 5 min
17. Transfer the aqueous phase to a new tube by pressing pipette tip against wall of tube; avoid puncturing the Phasemaker gel
18. Add an equal volume of 100% EtOH (~160-180uL) to each sample and mix thoroughly ,vortex and pulse spin
19. 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)
20. Add 400ul Direct-zol RNA PreWash to the column and centrifuge @ 16,000rcf for 30 seconds. Discard the FT. Use the vacuum-trap system.
21. Repeat step 20
22. 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.
23. To elute RNA, add 6ul of RNase-Free Water directly to the column matrix and centrifuge @ 16,000rcf for 30 seconds.
24. Send ~1ul of Elutions for pico chip bioanalyzer analysis (send 1.5ul to make it easier to load the chip).
25. Stored the rest of RNA samples at -80C; The organic phase was also saved after separating Trizol elution on phasemaker. Could potentially use this fraction for protein precipitation.
RNA samples: elution from anti-ORF1 IP
1. MT302 regular buffer/no EDTA or MgCl #1
2. MT302 regular buffer/no EDTA or MgCl #2
3. MT302 regular buffer/no EDTA or MgCl #3
4. MT302 EDTA #1
5. MT302 EDTA #2
6. MT302 EDTA #3
7. MT302 MgCl #1
8. MT302 MgCl #2
9. MT302 MgCl #3
Bioanalyzer Result
Original file /Users/hjiang01/Box/Wiki_raw_data/Hua's raw data/anti-ORF1 IP with EDTA or MgCl2_080520/EukaryoteTotalRNANano20200805Hua.pdf