Extracting RNA from Dinn-1 infected with DiNV for RNASeq

Cells had been infected here and either flash frozen at day 0 (no infection), day 1, day 3, or day 5. Tested this out on a few samples at first.

Process was very similar to the fly extracts, except no homogenization with a pestle.

Samples extracted:

sample number treatment day
9 CCM 1
25 CCM 3
41 CCM 5
13 20ul P4 DiNV 1
29 20ul P4 DiNV 3
45 20ul P4 DiNV 5

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 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
9 CCM 1 20240514 126
25 CCM 3 20240514 146
41 CCM 5 20240514 154
13 20ul P4 DiNV 1 20240514 128
29 20ul P4 DiNV 3 20240514 140
45 20ul P4 DiNV 5 20240514 144

Sample 9 and 45 were run on an RNA tapestation, see results here

All sample info is here