Extracting RNA from D.innubila infected with DiNV for RNASeq
Flies had been infected here and either flash frozen at day 0 (no injection), day 1, day 3, or day 5. Tested this out on a few samples at first.
Samples:
| sample number | treatment | day | day extracted |
|---|---|---|---|
| 1 | none | 0 | 20240514 |
| 2 | none | 0 | 20240514 |
| 6 | CCM | 1 | 20240514 |
| 7 | CCM | 1 | 20240514 |
| 16 | CCM | 3 | 20240514 |
| 17 | CCM | 3 | 20240514 |
| 26 | CCM | 5 | 20240514 |
| 11 | 1 FFU P4 DiNV | 1 | 20240514 |
| 12 | 1 FFU P4 DiNV | 1 | 20240514 |
| 21 | 1 FFU P4 DiNV | 3 | 20240514 |
| 22 | 1 FFU P4 DiNV | 3 | 20240514 |
| 31 | 1 FFU P4 DiNV | 5 | 20240514 |
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, placed tubes in rack
- Homogenized each fly individually with a sterile new pestle each time
- Discarded pestle in hazardous waste
- Centrifuged tubes 12,000g for 10 min at 4C
- Transferred supernatant to new tubes
- Let samples incubate at room temp for 5 minutes
- 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 260ul 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 25ul 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 | 20240514 | 160 |
| 2 | none | 0 | 20240514 | 168 |
| 6 | CCM | 1 | 20240514 | 172 |
| 7 | CCM | 1 | 20240514 | 168 |
| 16 | CCM | 3 | 20240514 | 158 |
| 17 | CCM | 3 | 20240514 | 158 |
| 26 | CCM | 5 | 20240514 | 156 |
| 11 | 1 FFU P4 DiNV | 1 | 20240514 | 162 |
| 12 | 1 FFU P4 DiNV | 1 | 20240514 | 163 |
| 21 | 1 FFU P4 DiNV | 3 | 20240514 | 148 |
| 22 | 1 FFU P4 DiNV | 3 | 20240514 | 158 |
| 31 | 1 FFU P4 DiNV | 5 | 20240514 | 134 |
Sample 16 and 12 were run on an RNA tapestation, see results here
All sample info is here