Preparation of mRNA libraries for sequencing on an Illumina platform
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Day 1

Documentation

TruSeq Stranded mRNA Sample Prep Guide. This method makes a cDNA library of the polyadenylated mRNA in your eukaryotic samples of interest.

What you’ll need for Day 1

  • RNA purification beads (These are the oligo dT beads) (bring these to room temperature before use)
  • Bead washing buffer
  • Bead binding buffer
  • Fragment prime finish mix
  • First strand synthesis (in a brown tube)
  • Second strand marking mix
  • Resuspension buffer
  • RNase-free water
  • Magnetic plate for a 96-well tray
  • 96-well trays
  • 80% ethanol (make this fresh the day of use)
  • Superscript II (Invitrogen)
  • AMPureXP beads (Beckman-Coulter)

A few important comments before you start

- We use the Low Sample ‘LS’ protocol as we are typically working with fewer than 48 samples at a time
- The recommended starting amount of total RNA is 100 ng - 4 ug, but we usually try to stay away from the extreme ends of this spectrum.
- The lowest input of RNA we have successfully used in this protocol is 70 ng.
- Split this protocol into two days, stopping on the first day after you have double stranded cDNA. On second day, do the A-tailing, adapter ligation, PCR, cleanup, and quality control steps.
- For 12 samples, you will need approximately 6-7 hours on the first day, and 6-7 hours the second day.
- Keep all reagents on ice unless otherwise stated.
- We typically remove the reagents from storage during an incubation period in the previous step.
- Have the beads at room temperature at least 30 minutes prior to use.
- Aliquot the reagents that arrive in large quantities (such as the resuspension buffer and the bead washing buffer) into 2 mL Eppendorf tubes.
- We do not perform any of the in-line controls.
- We do not use the plate barcode stickers that come with the kit.
- Use only filter pipette tips and clean your area so it is free of RNases.
- Ensure that the AMPureXP beads are mixed well immediately prior to use.
- There are many mixing steps in this protocol - I frequently use a multi-channel pipette to help speed up this process, particularly if many libraries are being prepared.
- We use a Tapestation or a BioAnalyzer to assay the input RNA quality. Ribosomal integrity numbers of 7 and higher are preferred.
- We quantify the input RNA using a Qubit fluorometer.

Step 1: Isolate mRNA

  1. Dilute total RNA in RNase-free water to final volume of 50 ul. Do this in a 96-well PCR plate (even if you have a few samples).

  2. Add 50 uL RNA purification beads to each well. Gently pipette up and down 6 times. Seal the plate with an adhesive seal.

  3. Place the sealed plate in the thermal cycler. Select the mRNA denaturation program (65C for 5 min, 4C hold).

  4. Remove the plate when it reaches 4C and incubate the plate at room temperature for 5 minutes.

  5. Remove the adhesive seal and place the plate on the magnetic stand at room temperature for 5 minutes

  6. Remove and discard the supernatant from each well of the plate

  7. Remove the plate from the magnetic stand and wash the beads by adding 190 uL bead washing buffer in each well. Gently pipette up and down 6 times.

  1. Place the plate on the magnetic stand at room temperature for 5 minutes

  2. Remove and discard the supernatant from each well.

  3. Remove the plate from the magnetic stand.

  4. Add 50 uL Elution Buffer to each well and gently pipette up and down 6 times. Seal the plate with an adhesive seal.

  5. Place the plate in the thermal cycler. Choose the program “mRNA elute” (80C for 2 min, 25 C hold).

  6. Remove the plate when the thermal cycler reaches 25C. Place the plate on the bench at room temperature and remove the seal.

  7. Add 50 uL Bead Binding Buffer to each well. Gently pipette up and down 6 times. Incubate the plate at room temperature for 5 minutes.

  8. Place the plate on the magnetic stand at room. Incubate the plate at room temperature for 5 minutes.

  9. Place the plate on the magnetic stand at room temp for 5 minutes. Remove and discard the supernatant from each well.

  10. Wash the beads by adding 190 uL bead washing buffer. Gently pipette up and down 6 times to mix.

  11. Place the plate on the magnetic stand at room temp for 5 min. Remove and discard the supernatant from each well.

  12. Remove the plate from the magnetic stand and add 19.5 uL Fragment, Prime, Finish Mix to each well. Gently pipette up and down 6 times to mix.

  13. Seal the plate with an adhesive film. Place the sealed plate on the thermal cycler. Select the Elute2Frag program (94C for 8 min, 4C hold).

  1. Remove the plate once it reaches 4C, centrifuge briefly, and then proceed immediately through the next steps.

  2. Remove the adhesive seal and place the plate on a magnetic stand at room temperature for 5 min.

  3. Remove 17 uL supernatant from each well into corresponding wells in a new plate. This contains your purified mRNA.

Step 2: 1st strand cDNA synthesis

  1. Remove First Strand Synthesis Act D Mix from -20C and thaw at RT. Centrifuge this reagent at 600 x g for 5 sec.

  2. Add 50 ul SuperScript II to the First Strand Synthesis Act D Mix well. If you’re not going to use the entire well of mix, then add SuperScript at a ratio of 1 ul SuperScript for every 9 ul of First Strand reagent. For example, for 12 reactions, mix 90 ul with 10 ul of SS II. Excess mix can be stored in the -20C freezer.

  3. Add 8 ul of First Strand SuperScript mix to each well containing the mRNA.

  4. Place the plate in thermocycler and run the “Synthesize 1st Strand” program (25C for 10 min, 42C for 15 min, 70C for 15 min, hold at 4C).

Step 3: 2nd strand cDNA synthesis

  1. Add 20 uL second strand marking mix and 5 uL resuspension buffer to each well. Pipette up and down 6 times.

  2. Centrifuge the plate at 600 x g for 30 sec.

  3. Place plate in thermocycler and run the ‘2nd strand’ reaction - 16C for 1hr, with the lid set to 30C.

Step 4: Reaction clean-up

  1. When the reaction is complete, add 90 ul of well-mixed AMPure XP beads to each well containing 50 ul of double stranded cDNA. Incubate RT for 15 min.

  2. Place the plate on the magnet and let sit for 5 min.

  3. Remove and discard 135 ul per well, being careful not to disturb the beads.

  4. Wash beads on magnet by adding 190 ul of 80% EtOH to each well, without disturbing the beads incubate 30 sec at RT.

  5. Remove and discard the supernatant, repeat the wash step and remove the supernatant again. Let the plate stand on the magnet at RT for 15 min to dry.

  1. Add 17.5 ul of Resuspension Buffer to each well and pipette up and down 10 time to mix. Incubate for 2 min at RT.
  1. Place the plate on the magnetic stand for 5 minutes.

  2. Transfer 15 ul of the supernatant (contains the ds cDNA) to new a new plate.

Day 2

What you’ll need for Day 2

  • A-tailing mix
  • Resuspension buffer
  • Ligation mix
  • Index adapters
  • Stop ligation buffer
  • PCR primer cocktail
  • PCR master mix
  • Magnetic plate for a 96-well tray
  • 96-well trays
  • 80% Ethanol (make this fresh the day of use)
  • AMPureXP beads (Beckman-Coulter)

Step 5: Adenylate cDNA ends

  1. Add 12.5 ul of thawed A-tailing mix and 2.5 uL of resuspension buffer to each well. Pipette up and down 10 times.

  2. Carry out A-tailing by running the thermocycler ‘ATAIL70’ program.

Step 6: ligate adapters to ends

  1. Add 2.5 ul of resuspension buffer, 2.5 ul of ligation mix, and 2.5 ul of a unique RNA adapter index to each well, mix well by pipetting.
  1. Seal the plate and incubate at 30C in thermocycler for 10 min using the ‘LIGADAPTER’ program.

  2. Remove the plate from the thermocycler and add 5 ul of stop ligation buffer to each reaction, mix well by pipetting.

  3. Add 42 ul of AMPure XP beads to each reaction, mix well by pipetting, and incubate at RT for 15 min.

  4. Place on magnetic stand for 5 min at RT.

  5. With the plate on the magnetic stand, remove 79.5 ul of supernatant from each sample, being careful not to disturb the beads.

  6. With the plate on the magnetic stand, wash with 190 ul 80% EtOH for 30 sec, then remove the ethanol.

  7. Perform the wash step again.

  8. Use a p10 pipette to remove residual ethanol. Leave the plate on the stand to dry for 15 minutes.

  9. Remove the plate from the magnetic stand and add 52.5 ul of Resuspension Buffer to each well, and mix well by pipetting. Incubate at RT for 2 min

  10. Place the plate on magnet for 5 min.

  11. Recover 50 ul from each well and transfer to a new plate.

  12. Carry out a second cleanup round. Add 50 uL of mixed AMPure beads to each well, mix well, incubate for 15 min at RT.

  13. Place the plate on the magnetic stand for 5 min.

  14. With the plate on the magnetic stand, remove and discard 95 ul of supernatant from each sample.

  15. With the plate on the magnetic stand, wash the beads with 190 ul of 80% EtOH for 30 s, then remove the ethanol.

  16. With the plate on the magnetic stand, wash the beads again with 190 uL of 80% EtOH for 30 s, then remove the ethanol.

  17. With the plate on the magnetic stand, use a p10 pipette to remove residual EtOH, then let the plate air dry for 15 min.

  18. Resuspend beads in 22.5 ul of resuspension Buffer, mix well by pipetting, and incubate 2 min at RT.

  19. Transfer the plate to the magnetic stand and incubate for 5 min.

  20. Recover 20 ul from each well and transfer to a new plate.

Step 7: PCR amplify library

  1. Add 5 ul of PCR primer cocktail and 25 ul of PCR master mix to each well, place the plate in the thermocycler and run the “PCR” program.
  1. Add 50 ul of AMPure beads to each well and mix well by pipetting. Incubate 15 min at RT.

  2. Place the plate on the magnet for 5 min.

  3. Remove and discard 95 ul of supernatant from each well.

  4. Wash the beads 190 ul/well of 80% EtOH, wait 30s, remove the supernatant.

  5. Repeat the ethanol wash step.

  6. Remove residual ethanol with a p10 pipette, let the beads air dry for 15 minutes on the magnetic plate.

  7. Remove the plate from the magnetic stand, add 32.5 ul of resuspension buffer and mix well, by pipetting, and incubate for 2 min at room temperature.

  8. Place the plate back on the magnetic stand for 5 minutes.

  9. Remove 30 uL of supernatant from each sample and move it into a new plate. This is the final cDNA product.