Preparing Libraries from RNA from D. innubila flies Infected with DiNV Set 2

Using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®. I had previously aliquoted out all the reagents into single-use tubes for 2 sets of 8 sample reactions, 2 sets of 21 sample reactions, and 2 sets of 16 sample reactions.

This lib prep kit is used in conjunction with NEBNext® Poly(A) mRNA Magnetic Isolation Module for mRNA isolation.

1000ng (1ug) input RNA was used for each sample. All sample info can be found here.

Sample prep

  • Thawed RNA on ice
  • Wiped down whole bench and all equipment with RNase away
  • Prepared samples in strip tubes:
Sample ul RNA for 1000ng ul nuclease free water to 50ul
16 6.3 43.7
17 6.3 43.7
12 6.1 43.9
22 6.3 43.7
3 6.3 43.7
4 6.3 43.7
8 5.5 44.5
9 5 45
18 5 45
19 8.3 41.7
27 8.3 41.7
28 8.3 41.7
29 8.3 41.7
13 7.7 42.3
14 7.7 42.3
24 9 41
32 9 41
33 5 45
34 9 41

mRNA capture and 1st Strand

  • Prepared 1st strand reaction buffer on ice
  • Using an 8 sample aliquot
  • Thawed reaction buffer and random primers on ice
  • Vortexed and spun them down
  • Prepped mix:
    • 8ul 1st strand buffer * 22 = 176ul
    • 2ul random primers * 22 = 44ul
    • 10ul nuclease free water * 22 = 220ul
  • Pipette mixed buffer and kept on ice
  • Prepared dT beads:
    • 20ul oligo dT beads * 22 = 440ul
    • 100ul RNA binding buffer * 22 = 2200ul
  • This can’t be done in 1 tube, so I made 3 and in each was:
    • 146.6ul oligo dT beads
    • 733.3ul RNA binding buffer
  • Pipetted 8 times to wash beads
  • Placed on magnet rack until clear
  • Removed supernatant
  • Removed tube from rack
  • Added 733.3ul 2X binding buffer and pipette mixed 6 times
  • Placed on magnet rack until clear
  • Removed supernatant
  • Removed tube from rack
  • Added 366.5ul 2X binding buffer to beads and pipette mixed 6 times
  • Added 50ul of bed mixture to each sample tube and pipette mixed 6 times (the 1000ng samples)
  • Placed samples in thermocycler 65C for 5 min then to 4C hold - NRNA1 lid set to 80C
  • Removed tubes
  • Pipette mixed samples 6x
  • Incubated samples at room temp for 5 minutes
  • Placed samples on magnet rack
  • Removed supernatant
  • Removed tube from rack
  • Added 200ul wash buffer to each tube and pipette mixed
  • Placed tubes on mag rack
  • Removed supernatant when clear
  • Removed supernatant
  • Removed tube from rack
  • Added 200ul wash buffer to each tube and pipette mixed
  • Placed tubes on mag rack
  • Removed supernatant when clear
  • Removed tube from rack
  • Added 50ul tris buffer (from kit) to each sample and pipette mixed
  • Placed samples in thermocycler 80C for 2 min then 25C hold - NRNA2 lid set to 105
  • Removed tubes
  • ADded 50ul RNA binding buffer to each tube and pipetted 6x
  • Incubated tubes at rom temp for 5 min
  • Placed tubes on mag rack
  • Discarded supernatant
  • Removed tube from rack
  • Added 200ul wash buffer to each tube and pipette mixed
  • Spun down tubes quickly
  • Placed tubes on mag rack
  • Removed supernatant when clear
    • Spun down tubes again
    • Placed back on rack and removed any leftover supernatant
  • Removed tube from rack
  • Added 11.5ul 1st strand buffer mix and resuspended the beads in each tube
  • Placed tubes in thermocycler 15 min at 94C then 4C hold - NRNA3 lid set to 105
  • Made a new set of strip tubes
  • Thawed strand specific reagent and 1st strand enzyme on ice
  • Took samples out before it got to 4C and placed on ice to cool for 1 min
  • Spun down tubes quickly then placed on the magnet
  • Quickly transferred 10ul of supernatant to the new set of strip tubes
  • Placed tubes on ice
  • Made 1st strand synthesis reaction mix on ice:
    • 8ul strand specific reagent * 22 = 176ul
    • 2ul 1st strand enzyme * 22 = 44ul
    • Pipette mixed
  • Added 10ul 1st strand reaction mix to each sample and pipette mixed
  • Placed tubes in thermocycler 1st strand reaction program - NRNA4 lid set to 105:
    • 10min at 25C
    • 15min at 42C
    • 15min at 70C
    • Hold at 4C
  • Afterwards placed tubes on ice
  • Made sure to keep PCR machine lid open after this program so it could cool

2nd Strand Synthesis

  • Thawed 2nd strand synthesis buffer and 2nd strand enzyme on ice
  • Prepared 2nd strand master mix:
    • 8ul 2nd strand synthesis buffer * 22 = 176ul
    • 4ul 2nd strand enzyme * 22 = 88ul
    • 48ul nuclease free water * 22 = 1056ul
  • Pipette mixed and kept on ice
  • Added 60ul mix to samples on ice and pipette mixed 10x
  • Placed samples in thermocycler 1 hour at 16C - NRNA5 lid set to OFF!

Bead clean after 2nd strand synthesis

Note: used the multichannel for these

  • Warmed and resuspended Ampure XP beads to room temp with gentle swirling
  • Prepared fresh 80% ethanol
  • After program, placed samples on bench
  • Added 144ul Ampure beads to each tube (1.8x) and pipette mixed 10x
  • Placed tubes on the orbital shaker at 150rpm for 10 minutes
  • Placed tubes on the magnet rack, then placed the rack on the orbital shaker for at least 10 minutes
    • the volume is really high here so it takes a long time for the beads to come to the magnet
  • Remove the clear supernatant (over 200ul)
  • Add 200ul 80% ethanol to each tube still on the rack avoiding the beads
  • Remove the ethanol
  • Add another 200ul 80% ethanol to each tube still on the rack avoiding the beads
  • Remove all ethanol, go back in after the p200 with a p20 and remove any residual liquid
  • Look at tubes and remove any droplets of ethanol present on the sides of the tubes with a pipette tips
  • Resuspend beads off the magnet in 53ul of 0.1X TE buffer
  • Incubate the tubes on the shaker at 150rpm for 5 min
  • Make a new set of strip tubes
  • Place tubes on the magnet
  • Remove the clear supernatant (50ul) to the new set of tubes
  • Place samples on ice

End Prep

  • Thawed end prep buffer and end prep enzyme on ice
    • Note that the buffer had a lot of precipitate, I had just warmed it to room temp and mixed it a lot before placing on ice. This could be because the kit is expired
  • Prepared end prep master mix on ice:
    • 7ul end prep buffer * 22 = 154ul
    • 3ul end prep enzyme * 22 = 66ul
  • Pipette mixed and kept on ice
  • Added 10ul end prep mix to each sample
  • Pipette mixed each tube 10x with 50ul
  • Placed tubes in thermocycler NRNA6 lis set to 105:
    • 30 min at 20C
    • 30 min at 65C
    • 4C hold
  • Afterwards, placed samples on ice
  • You must either cool the lid down after this or use another thermocycler because the next program needs the lid at 20C

Adapter Ligation

  • Thawed NEB next adapter, adapter buffer, ligation enhancer, ligation mix, and USER enzyme on ice
  • Diluted NEB next adapter by 5 (based on kit recommendations for 1ug input)
    • I need 55ul total of adapter
    • 11ul NEBNext adapter
    • 44ul adapter buffer
    • Vortexed and spun down
  • Made adapter ligation mix on ice:
    • 1ul ligation enhancer * 22 = 22ul
    • 30ul ligation mix * 22 = 660ul (this is very viscous)
    • Pipette to mix (very important, DNA ligase cannot be vortexed)
  • Added 31ul ligation mix to each sample
  • Added 2.5ul diluted adapter to each sample
  • Pipette mixed each sample with 80ul 10 times (viscous)
  • Placed in thermocycler 20C for 15 min - NRNA7 lid set to OFF!
  • Take out tubes
  • Add 3ul USER enzyme to each tube
  • Pipette mix with 80ul
  • Place in thermocycler 37C for 15 min - NRNA8 - lid set to 45C

Bead Purification 2

Note: used the multichannel for these

  • Took tubes out of thermocycler to bench
  • Added 87ul beads to each tube and pipette mixed 10x
  • Placed tubes on the orbital shaker at 150rpm for 10 minutes
  • Placed tubes on the magnet rack, then placed the rack on the orbital shaker for some time if needed
    • the volume is still high here and may need shaking
  • Remove the clear supernatant (over 200ul)
  • Add 200ul 80% ethanol to each tube still on the rack avoiding the beads
  • Remove the ethanol
  • Add another 200ul 80% ethanol to each tube still on the rack avoiding the beads
  • Remove all ethanol, go back in after the p200 with a p20 and remove any residual liquid
  • Look at tubes and remove any droplets of ethanol present on the sides of the tubes with a pipette tips
  • Resuspend beads off the magnet in 17ul of 0.1X TE buffer
  • Incubate the tubes on the shaker at 150rpm for 5 min
  • Make a new set of strip tubes
  • Place tubes on the magnet
  • Remove the clear supernatant (15ul) to the new set of tubes
  • Place samples on ice

Index Amplification

  • During the bead clean, thawed the index place on ice, then spun it down in the large centrifuge
  • Index pairs for each sample had already been planned, see this spreadsheet
  • Thawed Q5 mix on ice, vortexed, and spun down
  • Added 25ul Q5 mix to each sample
  • Added 10ul of the planned index to each tube - plate needs to be pierced with pipette tip to get into single use wells
  • Pipette mixed each tube with 40ul and spun down tubes
  • Placed tubes in thermocycler NRNA9 lid at 105:
    • 98C 30 sec
    • 98C 10 sc
    • 65C 75 sec
    • 65C 5 min
    • 4C hold
    • Note that italic lins were cycled 10 times (this was later changed to 9)

Bead Clean 3

Note: used the multichannel for these

  • Took tubes out of thermocycler to bench
  • Added 45ul beads (0.9x) to each tube and pipette mixed 10x
  • Placed tubes on the orbital shaker at 150rpm for 10 minutes
  • Placed tubes on the magnet rack and wait for it to become clear
  • Remove the clear supernatant (over 200ul)
  • Add 200ul 80% ethanol to each tube still on the rack avoiding the beads
  • Remove the ethanol
  • Add another 200ul 80% ethanol to each tube still on the rack avoiding the beads
  • Remove all ethanol, go back in after the p200 with a p20 and remove any residual liquid
  • Look at tubes and remove any droplets of ethanol present on the sides of the tubes with a pipette tips
  • Resuspend beads off the magnet in 23ul of 0.1X TE buffer
  • Incubate the tubes on the shaker at 150rpm for 5 min
  • Make a new set of strip tubes
  • Place tubes on the magnet
  • Remove the clear supernatant (21ul) to the new set of tubes
  • Place samples on ice
  • I immediately qubitd the samples:

HS DNA Qubit

Sample ng/ul
16 35.2
17 38.7
12 28.8
22 21.5
3 36.8
4 52.1
8 28
9 28.4
18 22.5
19 27.4
27 23.8
28 23.8
29 23.4
13 16.7
14 16.8
23 19.6
24 24.9
32 17.9
33 12.1
34 20.9

Samples were frozen at -20 until pooling