Sequencing Library Preparation: RNA-Seq

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RNA-Seq Library Prep Notes

  • This Protocol assumes you have final samples of purified Total RNA in dH2O or TE buffer as per Total RNA Extraction
  • The end goal of this Protocol is a finalized bar-coded cDNA sample suitable for Illumina Sequencing
  • All Buffer's and Components besides the original Total RNA sample are from Illumina Tru-Seq RNA Library Preparation Kit (LS or Low Sample)
  • Recommended Starting amount is 0.1 - 0.4 ug of Total RNA (dilute sample as necessary)
  • Prepare a fresh solution of 80% Ethanol each day using RNase Free H2O and 100% Ethanol (use a sealed bottle, or one marked for RNA use)


Note: Unless otherwise noted, ALL materials used in this protocol must be RNase free including Glassware (heat treated) unitl AFTER the synthesis of the 2nd strand of cDNA.

Day 1 (~5 hours)

Purification of mRNA from Total RNA sample (~1.5 hours)

Pre-Preparation for mRNA Purification

  • Remove the following reagents from the -200C Freezer and thaw on ice:
Bead Binding Buffer
Bead Washing Buffer
Elution Buffer
Elute, Prime, Fragment Mix
RNA Purification Beads
  • Remove RNA Purification Beads from storage and let stand for 30 min @ RT before use
  • After thawing solutions vortex and briefly centrifuge to collect droplets

Purification of mRNA from Total RNA sample

  1. Aliquot a volume of your samples containing 0.1-0.4 ug of Total RNA into a new sterile RNAase Free 0.2mL PCR tubes and label accordingly
  2. Dilute each sample of Total RNA with RNAase free water to a final volume of 50uL
  3. Thoroughly re-suspend RNA Purification Beads by vortexing
  4. Add 50uL of RNA purification Beads to your samples, and mix by pipetting volume up and down six (6) times
  5. Close and seal the PCR tube
  6. Heat the samples for 5 min @ 650C and then cool to 40C in a thermo-cycler
  7. Remove samples from the thermo-cycler and allow them to sit @ RT for 5 min
  8. Move samples to the Magnetic Stand and allow them to sit @ RT for at least 5 min to allow for separation of the beads and supernatant
  9. Carefully pipette off and discard the supernatant from your samples (See Note below)
  10. Remove samples from the Magnetic Stand and wash beads with 200uL of Bead Washing Buffer and mix by pipetting up and down six (6) times, being sure to re-suspend the beads
  11. Return samples to the Magnetic Stand and allow them to to sit @ RT for 5 min
  12. Carefully pipette off and discard the supernatant from your samples (See Note below)
  13. Remove samples from the Magnetic Stand and wash beads with 50uL of Elution Buffer and mix by pipetting up and down six (6) times, being sure to re-suspend the beads
  14. Transfer samples to new (RNase free) 0.2mL PCR tubes and label accordingly
  15. Place samples in the thermo-cycler for 2 min @ 800C then cool to 250C
  16. Remove samples from the thermo-cycler and add 50uL of Bead Binding Buffer and mix by pipetting up and down six (6) times
  17. Incubate samples @ RT for 5 min
  18. Return samples to the Magnetic Stand and allow them to to sit @ RT for 5 min
  19. Carefully pipette off and discard the supernatant from your samples (See Note below)
  20. Remove samples from the Magnetic Stand and add 19.5uL of Elute, Prime, Fragment Mix and pipette up and down six (6) times, being sure to re-suspend the beads
  21. Place samples in the thermo-cycler for 8 min @ 940C then bring to 40C
  22. After samples reach 40C remove them from the thermo-cycler and centrifuge briefly to collect droplets


Note The RNA Purification Beads should form a ring in the PCR tube near the top of edge of the magnet. Be careful not to disturb this ring when pipetting off supernatant.


cDNA Synthesis (~3.5 hours)

Pre-Preparation for First Strand cDNA Synthesis

  • Thaw one tube of First Strand Master Mix on ice
  • After thawing vortex and briefly centrifuge to collect droplets
  • Pre-program the thermo-cycler with the following program
Pre-heat lid to 1000C
250C for 10 min
420C for 50 min
700C for 15 min
40C holding temp


First Strand cDNA Synthesis (~1.5 hours)

  1. Return samples to the Magnetic Stand and allow them to sit for 5 min @ RT
  2. Carefully pipette off 17uL of the supernatant and transfer it to a new 0.2mL PCR tube (See Note below)
  3. Place the remaining sample in the -200C freezer for future use
  4. To each sample add 7.2uL of First Strand Master Mix and 0.8uL of Super Script II Reverse Transcriptase and pipette up and down six (6) times
  5. Place samples in the thermo-cycler and run the above program
  6. After samples reach 40C remove them from the thermo-cycler and proceed to Second Strand cDNA Synthesis


Pre-Preparation for Second Strand cDNA Synthesis

  • Thaw the following reagents on ice
Second Strand Master Mix
Re-suspension Buffer
  • Remove AMPure Beads from storage and let stand for 30 min @ RT before use
  • After thawing solutions vortex and briefly centrifuge to collect droplets


Second Strand cDNA Synthesis (~2.0 hours)

  1. To each sample add 25uL of Second Strand Master Mix and pipette up and down six (6) times to mix
  2. Close and seal the sample tubes
  3. Place samples in the thermo-cycler @ 160C for 1 hour
  4. Remove samples from the thermo-cycler and alow them to come to RT on the bench-top
  5. Transfer samples into a new 0.2uL PCR tube
  6. To each sample add 90uL of AMPure XP beads and pipette up and down six (6) times to mix
  7. Return samples to the Magnetic Stand and allow them to sit for 5 min @ RT
  8. Carefully pipette off and discard the supernatant (See Note below)
  9. Leave the samples on the Magnetic stand and wash with 200uL of freshly prepared 80% Ethanol, being careful not to disturb the beads
  10. Allow the wash to sit for 30 seconds, and then carefully pipette off and discard the supernatant (See Note below)
  11. Repeat steps 9 + 10 above two (2) additional times for a total of three (3) 80% Ethanol washes
  12. Allow the samples to stand on the Magnetic Rack for 15 min with their tops open (to allow residual wash to evaporate)
  13. Remove samples from the Magnetic Stand and add 52.5uL of Re-suspension Buffer and pipette solution up and down ten (10) times to mix
  14. Allow samples to sit @ RT for 2 min
  15. Return samples to the Magnetic Stand and allow them to sit for 5 min @ RT
  16. Carefully pipette off 50uL of the supernatant and and transfer it to a new 0.2mL PCR tube (See Note below)


Note The AMPure Beads should form a brown ring in the PCR tube near the top of edge of the magnet. Be careful not to disturb this ring when pipetting off supernatant

Note 2 Samples can safely be stored at -200C after this step


Day 2 (~5.5 hours)

End Repair (~2 Hours)

Pre-Preparation for End Repair

  • Thaw the following reagents on ice
10x Re-suspension Buffer
End Repair Mix
  • Remove AMPure Beads from storage and let stand for 30 min @ RT before use
  • After thawing solutions vortex and briefly centrifuge to collect droplets


End Repair

  1. Too each sample add 10uL of 10x Re-suspension Buffer and 40uL of End Repair Mix (final volume of 100uL)
  2. Incubate all samples in a preheated thermo-cycler @ 300C for 30 min
  3. Remove samples from the thermo-cyler and add 160uL of AMPure Beads and pipette solution up and down ten (10) times to mix
  4. Allow samples to sit @ RT for 15min
  5. Place samples in the Magnetic Stand and allow them to sit for 15 min
  6. Carefully pipette off and discard the supernatant (See Note below)
  7. Leave the samples on the Magnetic stand and wash with 200uL of freshly prepared 80% Ethanol, being careful not to disturb the beads
  8. Allow the wash to sit for 30 seconds, and then carefully pipette off and discard the supernatant (See Note below)
  9. Repeat Steps 6-7 above for a total of two (2) 80% Ethanol washes
  10. After removing the second wash, allow samples to air dry @ RT for 15 min
  11. Remove samples from the Magnetic Stand and re-suspend beads in 17.5uL of re-suspension buffer and pipette up and down gently (10x) to mix
  12. Incubate samples at RT for 2min
  13. Return samples to the Magnetic Stand and allow them to sit for 5 min @ RT
  14. Carefully pipette off 15uL of the supernatant and and transfer it to a new 0.2mL PCR tube (See Note below)


Note The AMPure Beads should form a brown ring in the PCR tube near the top of edge of the magnet. Be careful not to disturb this ring when pipetting off supernatant.

Note2: Samples can be stored at -200C for a few days at this stage if necessary


A-Overhang Addition (~1 hour)

Pre-Preparation for A-Overhang Addition

  • Remove samples from the -200C freezer and thaw on ice
Re-suspension buffer
A-tailing Mix
  • After thawing solutions vortex and briefly centrifuge to collect droplets


A-Overhang Addition

  1. To each sample add 2.5uL of Re-suspension buffer and 12.5uL of A-tailing Mix
  2. Gently pipette samples up and down (10x) to mix
  3. Incubate samples in a pre-heated Thermo-Cycler @ 370C for 30min


Note During thermo-cycler incubation, thaw and prepare Illumina Adapters


Illumina Adapter Ligation (~2.5 Hours)

Pre-Preparation for Illumina Adapter Ligation

  • Thaw the following reagents on ice
Illumina Tru-Seq Adaptors (only those you will be using)
Stop Ligation Buffer
  • After thawing solutions vortex and briefly centrifuge to collect droplets
  • Remove AMPure Beads from storage and let stand for 30 min @ RT before use

NOTE DO NOT thaw Ligation Mix. Leave in storage @ -200C until use, then immediately return to storage


Illumina Adapter Ligation

  1. To each sample add 2.5uL of Re-suspension Buffer, 2.5uL of Ligation Enzyme Mix, and 2.5uL of the Appropriate Illumina Adapter
  2. Incubate samples in preheated Thermo-Cycler @ 300C for 10 min
  3. Add 5uL of Stop Ligation Buffer to each sample and pipette solution up and down ten (10) times to mix
  4. Add 42.5uL of AMPure Beads and pipette solution up and down ten (10) times to mix
  5. Allow samples to sit @ RT for 15 min
  6. Place samples in the Magnetic Stand and allow them to stand @ RT for 5 min
  7. Carefully pipette off 80uL of the supernatant and discard (See Note below)
  8. Leave the samples on the Magnetic stand and wash with 200uL of freshly prepared 80% Ethanol, being careful not to disturb the beads
  9. Allow the wash to sit for 30 seconds, and then carefully pipette off and discard the supernatant (See Note below)
  10. Repeat Steps 8-9 above for a total of two (2) 80% Ethanol washes
  11. After removing the second wash, allow samples to air dry @ RT for 15 min
  12. Remove samples from the Magnetic Stand and add 52.5uL of Re-suspension buffer and pipette solution up and down ten (10) times to mix
  13. Incubate samples @ RT for 2 min
  14. Return samples to the Magnetic Stand and allow samples to stand @ RT for 5 min
  15. Carefully pipette off ~50uL of supernatant and conserve in a new 0.2mL PCR tube (See Note below)
  16. Remove samples from the Magnetic Stand
  17. To each sample add 50uL of AMPure Beads and pipette solution up and down ten (10) times to mix
  18. Return samples to the Magnetic Stand and allow them to sit for 5 min @ RT
  19. Carefully pipette off 95uL of supernatant and discard (See Note below)
  20. Leave the samples on the Magnetic Stand and wash with 200uL of freshly prepared 80% Ethanol, being careful not to disturb the beads
  21. Allow the wash to sit for 30 seconds, and then carefully pipette off and discard the supernatant (See Note below)
  22. Repeat Steps 20-21 above for a total of two (2) 80% Ethanol washes
  23. After removing the second wash, allow samples to air dry @ RT for 15 min
  24. Remove samples from the Magnetic Stand and add 22.5uL of Re-suspension buffer and pipette solution up and down ten (10) times to mix
  25. Allow samples to sit @ RT for 2 min
  26. Return the samples to the Magnetic Stand and and allow them to sit for 5 min @ RT
  27. Pipette off 20uL of the supernatant and transfer to a new 0.2mL PCR tube


Note The AMPure Beads should form a brown ring in the PCR tube near the top of edge of the magnet. Be careful not to disturb this ring when pipetting off supernatant.

Note2: Samples can be stored at -200C for a few days at this stage if necessary


Day 3 (~4 hours)

Gel Electrophoresis

Pre-Preparation for Gel Electrophoresis

  • Prepare an appropriate volume of TAE buffer
  • Prepare an appropriate volume of 2% Agarose Gel solution in TAE buffer using the low-range ultra-agarose gel
  • Cast an appropriate sized Gel w/ your 2% Agarose solution, be sure to cast wells large enough to hold ~25uL


Size Selection by Gel Electrophoresis

  1. Starting with the second lane, load the entirety of each sample into a single well, being sure to skip a lane between samples and to leave the last well empty
  2. Load 5uL of Fermentas 1KB Plus Ladder into each empty well on either side of your samples
  3. Run gel for 120 min at 100V and check for adequate band separation
  4. Locate and cut out gel plugs of the appropriate size for your sample (fragment size + adapter length x2) under UV Light (see Note below)
  5. Use the ladders on each side of your samples to guide your cuts (see Note below)
  6. Immediately after removing a gel plug place in new 2.0mL epi-tubes and label (see Note below)
  7. Gel-purify the resulting gel plugs with the QIAGEN MiniElute Kit and elute samples in 25uL of EB Buffer


Note Only cut out one gel plug at a time as they are very easy to lose and / or confuse with one another

Note2 When purifying gel plugs dissolve at RT, not at 500C as called for in QIAGEN protocol sheet

Note3 Gel plugs or purified samples can be stored at -200C for a few days at this stage if necessary

Note4 Purified Samples can be safely stored indefinitely at -200C at this stage

Day 4 (~4 hours)

PCR Amplification of Library Samples

Pre-Preparation for PCR Amplification

  • Thaw the following reagents on ice
PCR Primer Cocktail
PCR Master Mix
  • After thawing solutions vortex and briefly centrifuge to collect droplets
  • Remove AMPure Beads from storage and let stand for 30 min @ RT before use
  • Pre-program the thermo-cycler with the following program


Pre-heat lid to 1000C
Step 1- 980C for 30seconds
Step 2- 980C for 10s
Step 3- 600C for 30s
Step 4- 720C for 30s
Repeat Steps 2 through 4 an additional fourteen times (14), for a total of fifteen (15) cycles
720C for 5min
40C holding temp

PCR Amplification

  1. To each sample add 5uL of PCR Primer Cocktail and 25uL of PCR Master Mix
  2. Close and seal the sample tubes and place in the thermo-cycler and run the above program
  3. Add 50uL of AMPure beads and and pipette solution up and down ten (10) times to mix
  4. Allow samples to sit @ RT for 15 min
  5. Place samples in the Magnetic Stand and allow them to stand @ RT for 5 min
  6. Carefully pipette off 95uL of supernatant and discard (See Note below)
  7. Leave the samples on the Magnetic Stand and wash with 200uL of freshly prepared 80% Ethanol, being careful not to disturb the beads
  8. Allow the wash to sit for 30 seconds, and then carefully pipette off and discard the supernatant (See Note below)
  9. Repeat Steps 7-8 above for a total of two (2) 80% Ethanol washes
  10. After removing the second wash, allow samples to air dry @ RT for 15 min
  11. Remove samples from the Magnetic Stand and add 32.5uL of Re-suspension buffer and pipette solution up and down ten (10) times to mix
  12. Incubate samples @ RT for 2 min
  13. Return the samples to the Magnetic Stand and and allow them to sit for 5 min @ RT
  14. Pipette off 30uL of the supernatant and transfer to a new 1.5mL epi-tube and label
  15. Validate library samples by running a small amount of the final sample on an agarose gel to check size of library smear


Note The AMPure Beads should form a brown ring in the PCR tube near the top of edge of the magnet. Be careful not to disturb this ring when pipetting off supernatant

Note2: Finalized library samples can be stored indefinitely at -200C indefinitely

Note3: Re-do gel purification step if validation shows prominent adapter band.



Adapted from the Illumina True-Seq RNA Sample Preparation Guide by Michael Seymour and Kelsey Lynch, 2013

See: http://www.illumina.com/products/truseq_dna_sample_prep_kits.ilmn