Extracting RNA from D.innubila infected with DiNV for RNASeq Set 2
Flies had been infected here and either flash frozen at day 0 (no injection), day 1, day 3, or day 5. This extraction worked well on the first set of flies so I proceeded with all the others. I increased the end volume because the concentration was so high with the first set:
Samples:
| sample number | treatment | day | day extracted |
|---|---|---|---|
| 3 | none | 0 | 20240515 |
| 4 | none | 0 | 20240515 |
| 8 | CCM | 1 | 20240515 |
| 9 | CCM | 1 | 20240515 |
| 18 | CCM | 3 | 20240515 |
| 19 | CCM | 3 | 20240515 |
| 27 | CCM | 5 | 20240515 |
| 28 | CCM | 5 | 20240515 |
| 29 | CCM | 5 | 20240515 |
| 13 | 1 FFU P4 DiNV | 1 | 20240515 |
| 14 | 1 FFU P4 DiNV | 1 | 20240515 |
| 23 | 1 FFU P4 DiNV | 3 | 20240515 |
| 24 | 1 FFU P4 DiNV | 3 | 20240515 |
| 32 | 1 FFU P4 DiNV | 5 | 20240515 |
| 33 | 1 FFU P4 DiNV | 5 | 20240515 |
| 34 | 1 FFU P4 DiNV | 5 | 20240515 |
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 270ul 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 35ul 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 |
|---|---|---|---|---|
| 3 | none | 0 | 20240515 | 160 |
| 4 | none | 0 | 20240515 | 160 |
| 8 | CCM | 1 | 20240515 | 180 |
| 9 | CCM | 1 | 20240515 | 200 |
| 18 | CCM | 3 | 20240515 | 200 |
| 19 | CCM | 3 | 20240515 | 120 |
| 27 | CCM | 5 | 20240515 | 120 |
| 28 | CCM | 5 | 20240515 | 120 |
| 29 | CCM | 5 | 20240515 | 120 |
| 13 | 1 FFU P4 DiNV | 1 | 20240515 | 130 |
| 14 | 1 FFU P4 DiNV | 1 | 20240515 | 130 |
| 23 | 1 FFU P4 DiNV | 3 | 20240515 | 130 |
| 24 | 1 FFU P4 DiNV | 3 | 20240515 | 110 |
| 32 | 1 FFU P4 DiNV | 5 | 20240515 | 110 |
| 33 | 1 FFU P4 DiNV | 5 | 20240515 | 200 |
| 34 | 1 FFU P4 DiNV | 5 | 20240515 | 110 |
Note that for some samples the volume was increased to 55ul of water because originally the qubit was too high to read.
Samples 9, 28, and 32 were run on an RNA tapestation, see results here
All sample info is here