- Day 1
- Documentation
- What you’ll need for Day 1
- A few important comments before you start
- Step 1: Isolate mRNA
- Step 2: 1st strand cDNA synthesis
- Step 3: 2nd strand cDNA synthesis
- Step 4: Reaction clean-up
- Day 2
- What you’ll need for Day 2
- Step 5: Adenylate cDNA ends
- Step 6: ligate adapters to ends
- Step 7: PCR amplify library
- Sequencing Guidelines
- Normalize and Pool
- Setup Run in Basespace
- Loading the Sequencer
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
The following items are included in the Illumina kit. We recommend kits be stored as received in the original manufacterer’s boxes and not unpacked to organize in a reagent box. When kits go bad, it is much easier to contain the damage if reagents were stored as received.
Reagent | Location | Box Name | Action |
---|---|---|---|
A-tailing mix | -20C Freezer | TruSeq Strnd RNA Core LP Box 1 | Thaw at room temp |
Resuspension buffer | Aliquot from -20C freezer, aliquots stored in 4C fridge | TruSeq Strnd RNA Core LP Box 1 (bulk tube), TruSeq Stranded mRNA LP Box 1 (aliquots) | Thaw at room temp for 30 min (You will need 112.5 µL per sample for Day 2) |
Ligation Mix (LIG) | -20C Freezer | TruSeq Strnd RNA Core LP Box 1 | Store in freezer until immediately before use, and replace immediately after use |
Index Adaptors | -20C Freezer | TruSeq RNA CD Indexes | Thaw at room temp for 10 min, reseal and replace after use |
Stop Ligation Buffer | -20C Freezer | TruSeq Strnd RNA Core LP Box 1 | Thaw at room temp, centrifuge before use |
PCR Primer Cocktail (PPC) | -20C Freezer | TruSeq Stranded RNA Core LP Box 2 | Thaw at room temp, invert to mix, centrifuge (NO VORTEX) |
PCR Master Mix (PMM) | -20C Freezer | TruSeq Stranded RNA Core LP Box 2 | Thaw on ice, invert to mix, centrifuge (NO VORTEX) |
The following items are NOT included with the Illumina kit and must be purchased separately
Name | Location | Action |
---|---|---|
80% Ethanol | Bench | mix 200 proof EtOH and Milli-Q water to make fresh 80% EtOH each day (you will need 1200 µL per sample for day 2) |
AMPureXP beads (Beckman-Coulter) | 4C Fridge | Thaw at room temp for 30 min then vortex vigorously |
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
Do not store mRNA purification beads on ice! We have observed that the beads are very sensitive to cold temperatures. Beads stored at < 2degC may deteriorate, resulting in poor binding of mRNA, and therefore poor library prep that is heavily contaminated with excess adapter.
The TruSeq manual indicates that 200 uL should be used, however, the maximum volume of our 96-well plates is 200 uL, thus we use less so the wells do not overflow.
For degraded mRNA or mRNA isolated from FFPE samples, you may shorten the fragmentation time. See the Illumina TruSeq instruction manual for suggested times.
If RNA is degraded and you do not want to Fragment, place samples on thermal cycler at 65C for 5 mins, 4C hold. This will elute the mRNA from the beads without degrading the RNA.
Step 2: 1st strand cDNA synthesis
This step takes 40 minutes and is your first large break.
Step 3: 2nd strand cDNA synthesis
When the first strand synthesis is completed, immediately proceed to the second strand synthesis.
This is your second large break of the day
Step 4: Reaction clean-up
Use a p10 pipette to remove any residual ethanol from the bottom of the well.
The beads may be dry and you may have to pipette up and down more than 10 times until the beads are fully resuspended.
This is a safe-stopping place. You can cover the plate and store at -20C until you are ready to continue to the second day of the protocol.
Do not store your DNA at this stage for longer than a week. We usually perform the rest of the protocol the next day.
Day 2
What you’ll need for Day 2
The following items are included in the Illumina kit
Reagent | Location | Box Name | Action |
---|---|---|---|
A-tailing mix | -20C Freezer | TruSeq Strnd RNA Core LP Box 1 | Thaw at room temp |
Resuspension buffer | Aliquot from -20C freezer, aliquots stored in 4C fridge | TruSeq Strnd RNA Core LP Box 1 (bulk tube), TruSeq Stranded mRNA LP Box 1 (aliquots) | Thaw at room temp for 30 min (You will need 112.5 µL per sample for Day 2) |
Ligation Mix (LIG) | -20C Freezer | TruSeq Strnd RNA Core LP Box 1 | Store in freezer until immediately before use, and replace immediately after use |
Index Adaptors | -20C Freezer | TruSeq RNA CD Indexes | Thaw at room temp for 10 min, reseal and replace after use |
Stop Ligation Buffer | -20C Freezer | TruSeq Strnd RNA Core LP Box 1 | Thaw at room temp, centrifuge before use |
PCR Primer Cocktail (PPC) | -20C Freezer | TruSeq Stranded RNA Core LP Box 2 | Thaw at room temp, invert to mix, centrifuge (NO VORTEX) |
PCR Master Mix (PMM) | -20C Freezer | TruSeq Stranded RNA Core LP Box 2 | Thaw on ice, invert to mix, centrifuge (NO VORTEX) |
The following items are NOT included with the Illumina kit and must be purchased separately
Name | Location | Action |
---|---|---|
80% Ethanol | Bench | mix 200 proof EtOH and Milli-Q water to make fresh 80% EtOH each day (you will need 1200 µL per sample for day 2) |
AMPureXP beads (Beckman-Coulter) | 4C Fridge | Thaw at room temp for 30 min then vortex vigorously |
Step 5: Adenylate cDNA ends
This step takes 35 minutes and is the first break of the day.
Step 6: ligate adapters to ends
Remove the ligation mix immediately before use and return to -20C storage immediately after use.
Individual adapter index tubes have been moved to strip tubes to make it easier to add the unique indexes to the wells with a multichannel pipette.
Make sure to record which sample received which index adapter number.
Step 7: PCR amplify library
This is another break for 30 minutes.
Congratulations! You’re done!…well, almost
Perform quality control steps - we use a Tapestation to determine the library size and qubit to quantify the libraries.
We recommend sequencing the libraries within 3 weeks.
Sequencing Guidelines
Normalize and Pool
Setup Run in Basespace
Loading the Sequencer
- The next step is to dilute and denature the prepared libraries. Illumina’s general guidelines for this on the NextSeq can be found here.
- Illumina’s system guide for the NextSeq, which covers the sequening workflow, can be found here.
- Your final loading concentration should be 1.3 - 1.8 pM, with most pools loaded at 1.4 or 1.5 pM.