Infecting Male D. innubila with 1FFU P4 DiNV For RNA
Infected flies needed for time course for RNASeq, flies for day 0, 1, 3, and 5. Males only and each sample is going to be a combo of 5 flies together (reduce any individual variation)
Dilute DiNV
- Using virus stock tube 38, P4 DiNV
- First diluted the virus for 6 FFU delivery in 27.6nl
- 24.3ul of stock
- 2.42ul of cell culture medium
- pipette mixed
- Then I diluted the 6 FFU solution by 6X to get a solution to deliver 1 FFU
- 5ul diluted virus
- 25ul cell culture medium
- pipette mixed
Infection
- Flies were 5-7 days old for this
- Only males were used
- 24-28 flies were put in each vial after injection to make freezing easier, because each sample needs to be 5 flies in one
- Day 0 flies were not injected with anything. They were put on CO2, pushed into 1.5mL tubes, then flash frozen in LN2:
| sample number | number of flies | treatment | day |
|---|---|---|---|
| 1 | 5 | NA | 0 |
| 2 | 5 | NA | 0 |
| 3 | 5 | NA | 0 |
| 4 | 5 | NA | 0 |
| 5 | 5 | NA | 0 |
- For the injected flies, the process followed the nanoinjection protocol
- Because of the large number of flies needed per vial, to sets of infections were done per vial so there wasn’t too much time on CO2 for any given fly
| vial | treatment | freezing day | time | time on | N# | total N# |
|---|---|---|---|---|---|---|
| 1 | CCM | 1 | 3:45 | 6min | 15 | 27 |
| 3:51 | 6min | 12 | ||||
| 2 | CCM | 3 | 3:57 | 6min | 13 | 25 |
| 4:03 | 6min | 12 | ||||
| 3 | CCM | 5 | 4:11 | 6min | 14 | 28 |
| 4:17 | 7min | 14 | ||||
| 4 | P4 DiNV | 1 | 4:28 | 8min | 15 | 27 |
| 4:36 | 6min | 12 | ||||
| 5 | P4 DiNV | 3 | 4:42 | 6min | 12 | 24 |
| 4:48 | 6min | 12 | ||||
| 6 | P4 DiNV | 5 | 4:54 | 6min | 14 | 28 |
| 5:02 | 6min | 14 |
On the day specified, the flies were placed on CO2, 5 individuals were put into each 1.5mL tube, and the tubes were flash frozen in LN2 and placed at -80.
All samples:
| sample number | number of flies | treatment | day |
|---|---|---|---|
| 1 | 5 | NA | 0 |
| 2 | 5 | NA | 0 |
| 3 | 5 | NA | 0 |
| 4 | 5 | NA | 0 |
| 5 | 5 | NA | 0 |
| 6 | 5 | CCM | 1 |
| 7 | 5 | CCM | 1 |
| 8 | 5 | CCM | 1 |
| 9 | 5 | CCM | 1 |
| 10 | 5 | CCM | 1 |
| 11 | 5 | 1 FFU P4 DiNV | 1 |
| 12 | 5 | 1 FFU P4 DiNV | 1 |
| 13 | 5 | 1 FFU P4 DiNV | 1 |
| 14 | 5 | 1 FFU P4 DiNV | 1 |
| 15 | 5 | 1 FFU P4 DiNV | 1 |
| 16 | 5 | CCM | 3 |
| 17 | 5 | CCM | 3 |
| 18 | 5 | CCM | 3 |
| 19 | 5 | CCM | 3 |
| 20 | 5 | CCM | 3 |
| 21 | 5 | 1 FFU P4 DiNV | 3 |
| 22 | 5 | 1 FFU P4 DiNV | 3 |
| 23 | 5 | 1 FFU P4 DiNV | 3 |
| 24 | 5 | 1 FFU P4 DiNV | 3 |
| 25 | 4 | 1 FFU P4 DiNV | 3 |
| 26 | 5 | CCM | 5 |
| 27 | 5 | CCM | 5 |
| 28 | 5 | CCM | 5 |
| 29 | 5 | CCM | 5 |
| 30 | 5 | CCM | 5 |
| 31 | 5 | 1 FFU P4 DiNV | 5 |
| 32 | 5 | 1 FFU P4 DiNV | 5 |
| 33 | 5 | 1 FFU P4 DiNV | 5 |
| 34 | 5 | 1 FFU P4 DiNV | 5 |
| 35 | 5 | 1 FFU P4 DiNV | 5 |
Sample info can be found here