Extracting RNA from Dinn-1 infected with DiNV for RNASeq 2
Cells had been infected here and either flash frozen at day 0 (no infection), day 1, day 3, or day 5. The first set went well so I went ahead and did the rest of them. I also included 80ul of the P4 virus stock as a sample, this was to check that there was no RNA from any other virus in our stocks.
Process was very similar to the fly extracts, except no homogenization with a pestle.
Samples extracted:
| sample number | treatment | day | |
|---|---|---|---|
| 1 | none | 0 | 20240516 |
| 2 | none | 0 | 20240516 |
| 3 | none | 0 | 20240516 |
| 4 | none | 0 | 20240516 |
| 10 | CCM | 1 | 20240516 |
| 11 | CCM | 1 | 20240516 |
| 12 | CCM | 1 | 20240516 |
| 26 | CCM | 3 | 20240516 |
| 27 | CCM | 3 | 20240516 |
| 28 | CCM | 3 | 20240516 |
| 42 | CCM | 5 | 20240516 |
| 43 | CCM | 5 | 20240516 |
| 44 | CCM | 5 | 20240516 |
| 14 | 20ul P4 DiNV | 1 | 20240516 |
| 15 | 20ul P4 DiNV | 1 | 20240516 |
| 16 | 20ul P4 DiNV | 1 | 20240516 |
| 30 | 20ul P4 DiNV | 3 | 20240516 |
| 31 | 20ul P4 DiNV | 3 | 20240516 |
| 32 | 20ul P4 DiNV | 3 | 20240516 |
| 46 | 20ul P4 DiNV | 5 | 20240516 |
| 47 | 20ul P4 DiNV | 5 | 20240516 |
| 48 | 20ul P4 DiNV | 5 | 20240516 |
| P4 | P4 solution | NA | 20240516 |
Extraction
- Placed centrifuge at 4C
- Got dry ice from BioStore
- Prepared fresh 100% isopropanol and 75% ethanol
- Wiped down all surfaced and equipment with RNase Away, working in the fume hood, had set up both solid and liquid waste disposal containers
- Placed sample tubes on dry ice
- Added 500ul cold trizol reagent to each tube
- Pipette mixed samples to homogenize
- Incubated samples at room temp for 5 min
- Added 100ul of cold chloroform to each tube
- Inverted tubes by hand rapidly for 15 seconds
- Incubated tubes for 3 minutes at room temp
- Centrifuged tubes for 15 min at 12,000g at 4C
- There was phase separation in the tubes
- Removed the aqueous (top) phase to new tubes
- about 290ul for each tube
- Discarded organic phase in liquid waste then tube in solid waste
- Added 1ul of glycogen to each tube
- Added 250ul of 100% isopropanol to each tube
- This is supposed to be .5mL of isopropanol to every 1mL of trizol used, so because I used .5mL trizol, I used .25mL isopropanol
- Inverted tubes to mix
- Incubated tubes at room temp for 10 minutes
- Centrifuged tubes for 10 minutes at 4C 12,000g
- There was a visible pellet in each tube
- Removed the supernatant to hazardous waste
- Washed the pellet with 1mL 75% ethanol
- Inverted sample 2x
- Centrifuged 7,500g for 5 min at 4C
- Air dried the pellet for 5 minutes (don’t ovr dry!)
- Resuspended pellet in 30ul of nuclease free water from the DNase kit
- Incubated tubes in heat block at 55C for 10 minutes
- Moved directly to DNA removal
Invitrogen™ DNA-free™ DNA Removal Kit
- Need to remove any contaminating DNA
- Thawed reagents on ice, vortexed and spun down (not inactivation reagent)
- Added 2.5ul 10X DNase I buffer to each tube
- Added 1ul rDNase I to each tube
- Pipette mixed
- Incubated tubes on heat block at 37C for 20 min
- Resuspended DNase inactivation reagent by flicking
- Added 2.5ul DNase inactivation reagent to each tube
- Pipette mixed
- Incubated tubes for 2 min at room temp on orbital shaker
- Centrifuged tubes at 10,000g for 1.5 min at room temp
- Transferred the supernatant to new 1.5mL tubes
- Placed tubes on ice
- Immediately quantified samples, then afterwards froze them at -80
Qubit
Followed general qubit protocol, used HS RNA kit
| sample number | treatment | day | day extracted | qubit |
|---|---|---|---|---|
| 1 | none | 0 | 20240516 | 120 |
| 2 | none | 0 | 20240516 | 120 |
| 3 | none | 0 | 20240516 | 120 |
| 4 | none | 0 | 20240516 | 150 |
| 10 | CCM | 1 | 20240516 | 120 |
| 11 | CCM | 1 | 20240516 | 120 |
| 12 | CCM | 1 | 20240516 | 140 |
| 26 | CCM | 3 | 20240516 | 150 |
| 27 | CCM | 3 | 20240516 | 140 |
| 28 | CCM | 3 | 20240516 | 160 |
| 42 | CCM | 5 | 20240516 | 150 |
| 43 | CCM | 5 | 20240516 | 160 |
| 44 | CCM | 5 | 20240516 | 160 |
| 14 | 20ul P4 DiNV | 1 | 20240516 | 140 |
| 15 | 20ul P4 DiNV | 1 | 20240516 | 120 |
| 16 | 20ul P4 DiNV | 1 | 20240516 | 150 |
| 30 | 20ul P4 DiNV | 3 | 20240516 | 140 |
| 31 | 20ul P4 DiNV | 3 | 20240516 | 150 |
| 32 | 20ul P4 DiNV | 3 | 20240516 | 170 |
| 46 | 20ul P4 DiNV | 5 | 20240516 | 170 |
| 47 | 20ul P4 DiNV | 5 | 20240516 | 170 |
| 48 | 20ul P4 DiNV | 5 | 20240516 | 170 |
| P4 | P4 solution | NA | 20240516 | 71.8 |
Samples 10, 44, 30 and P4 were run on an RNA tapestation, see results here
All sample info is here