General LaCava lab protocol, Dec 10 2019
RNA extraction after cell powder IP
Materials
| Name | Company | Cat. nr. | Date received/made | Comments | ||||
|---|---|---|---|---|---|---|---|---|
| 0.5M Hepes, pH 7.4 | Nuclease free stock solution | |||||||
| 5M NaCl | Nuclease free stock solution | |||||||
| 10% Triton X-100 | Nuclease free stock solution | |||||||
| Protease inhibitor | Roche | # 05056489001 | Dissolved in nuclease free water for 100x stock solution | |||||
| RNAsin | Promega | # N2515 | Recombinant Ribonuclease inhibitor | |||||
| Phasemaker tubes | ||||||||
| Trizol | ||||||||
| EtOH | ||||||||
| Chloroform | ||||||||
| Direct-Zol RNA micro-prep kit | Zymo Research |
Steps
Precautions
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);
4. During spin, wash 10ul 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 (“Input”) for protein analysis, and optionally 35ul of supernatant for RNA prep; snap freeze RNA portion in Trizol in liquid nitrogen
6. Set up IP with 10ul of anti-ORF1 beads/sample (made on )
7. IP@ 4°C for 30’ (get tubes and Zymo columns ready while IP is going)
8. Save 10ul of flow-through (“FT”) for protein analysis and optionally 35ul of supernatant for RNA prep; snap freeze RNA portion in Trizol in liquid nitrogen
9. Wash 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
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. Add 25ul water and then 50ul Chloroform to the Phasemaker. Do this immediately before adding TriZol elutions or (perhaps safer still) immediately after transfering elutions to the Phasemaker tube. Adding Chloroform/water too far in advance may cause a failure. Not adding the water will cause the PhaseMaker to fail and 'swallow' the aqueous portion of the TriZol reagent mix.
15. Collect the elution from step 12 (“E”, pulse spin, place on magnet) and transfer to the Phasemaker tube
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, 4C (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
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)
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. Send ~1ul of Elutions for pico chip bioanalyzer analysis.
27. Optional: remainder saved for RNA-seq analysis ~4ul
28. All stored at -80C