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- Documentation
- A few important comments before you start
- Deplete rRNA
- STRANDED TOTAL RNA WITH RIBO-ZERO PLUS: Step 1: Hybridize Probes
- STRANDED TOTAL RNA WITH RIBO-ZERO PLUS MICROBIOME: Step 1: Hybridize Probes
- Step 2: Clean Up and Fragment RNA
- Generate cDNA
- Step 3: First strand cDNA synthesis
- Step 4: Second strand cDNA synthesis
- Step 5: cDNA clean-up
- Prepare and Amplify Libraries
- Step 6: Adenylate cDNA ends
- Step 7: Ligate Anchors
- Step 8: Clean Up Adapter-Ligated Fragments
- Step 9: Index and Amplify library
- Step 10: Clean Up Library
- Quality Checks
- Sequencing Guidelines
- Normalize and Pool
Documentation
Illumina's Stranded Total RNA Sample Prep Reference Guide. This method makes a cDNA library of all RNA molecules present in your sample after rRNA depletion.
Ribo-Zero Plus targets rRNA of human, rat, mouse, and bacteria for depletion. The success of this kit is dependent on the ability of Illumina’s rRNA depletion reagents to bind rRNAs in your target species. Check this list for compatibility of Ribo-Zero Plus depletion with your experiment.
This protocol can be modified slightly for the Stranded Total RNA Prep, Ligation with Ribo-Zero Plus Microbiome kit. THe microbiome kit is designed to remove rRNA from species most commonly found in the human microbiome. The alteration can be found in Step 1, but all other steps are the same.
A few important comments before you start
- This kit has a recommended range of 1–1000 ng purified total RNA input from high quality RNA samples (RIN ≥ 9). It optimized for 10–100 ng RNA input and our preferred use of this kit is high quality RNA at 100ng total input.
- Low-quality RNA (RIN ≥ 2) or FFPE (DV200 > 55%) samples can be used with this kit with 10–100 ng RNA input. Library performance can vary with lower input amounts and lesser quality RNA and we recommend using a different kit.
- This protocol can be performed in one day, or split into two or more days at the indicated safe stopping points.
- For 12 samples, you will need approximately 7.5 hours if completing the protocol in one day. This does not include QC steps before or after the protocol or sequencing. This estimate is based on an experienced user with multichannel pipette and vortexing bead cleanup steps. If prepping greater than 12 samples or the user is inexperienced or not using time-saving techniques, plan for more time and consider a two day prep.
- Keep all reagents on ice unless otherwise stated. Do NOT store any reagents from the fridge, especially beads, on ice.
- Have all beads at room temperature at least 30 minutes prior to use.
- Aliquot the reagents that arrive in large quantities (such as bead washing buffer) into 2 mL Eppendorf tubes.
- 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. Samples can also be mixed by sealing the plate and shaking as described in the Sample Prep Guide linked above.
- We use a Tapestation to assay the input RNA quality. RNA Integrity Numbers (RIN) of 7 and higher are preferred.
- We quantify the input RNA using a Qubit fluorometer.
Deplete rRNA
The following items are included in the Illumina kit. We recommend kits be stored as received in the original manufacturer’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.
This kit has 3 boxes of reagents in the -20C freezer, each labeled with a red dot saying "Total RNA". Make sure you are using the correct boxes and return all reagents to the box they came from. Do not mix reagents with other boxes.
| NAME | LOCATION | ACTION |
|---|---|---|
Elution Buffer (ELB) | -20°C Freezer (Ribo Zero Plus box, clear cap) | Thaw at RT, vortex and centrifuge briefly |
Depletion Probe Buffer (DB1) | -20°C Freezer (Ribo Zero Plus box, blue cap) | Thaw at RT, vortex and centrifuge briefly |
Depletion Probe Pool (DP1) | -20°C Freezer (Ribo Zero Plus box, blue cap) | Thaw at RT, vortex and centrifuge briefly |
Depletion Probe Microbiome (DPM) (for microbiome kit ONLY) | -20°C Freezer (alone in its own box) | Thaw at RT, vortex and centrifuge briefly |
RNA Depletion Buffer (RDB) | -20°C Freezer (Ribo Zero Plus box, yellow cap) | Thaw and keep on ice, vortex briefly before use |
RNA Depletion Enzyme (RDE) | -20°C Freezer (Ribo Zero Plus box, yellow cap) | Store until needed, flick to mix, centrifuge briefly |
Probe Removal Buffer (PRB) | -20°C Freezer (Ribo Zero Plus box, red cap) | Thaw at RT, vortex and centrifuge briefly |
Probe Removal Enzyme (PRE) | -20°C Freezer (Ribo Zero Plus box, red cap) | Store until needed, flick to mix, centrifuge briefly |
| NAME | LOCATION | ACTION | VOLUME |
|---|---|---|---|
Ampure XP Beads/Homemade beads | 4°C Fridge | Let stand at RT for 30 minutes to bring to RT, vortex and invert thoroughly before use | 174uL per sample for entire prep |
RNAClean XP Beads (Ampure beads can also be used, but RNAClean are certified free of RNases) | 4°C Fridge | Let stand at RT for 30 minutes to bring to RT, vortex and invert thoroughly before use | 60 uL per sample for entire prep |
Fresh 80% Ethanol (200 proof EtOH + MilliQ water) | RT Bench | Mix fresh each day. Low% EtOH negatively affects cleanups. | 1.6mL per sample for entire prep |
RNAse Free RT-PCR Grade Water | -20°C Freezer | Thaw at RT | As needed for diluting RNA input |
Magnetic Stand for 96 well plates | RT Bench |
STRANDED TOTAL RNA WITH RIBO-ZERO PLUS: Step 1: Hybridize Probes
Dilute 1-1000ng (typically 100ng) total RNA in nuclease-free water to a total volume of 11 uL. Do this in a 96-well PCR plate (if only prepping a few samples, the plate can be cut, but smaller than a half plate can be unstable on the magnet during cleanup steps). We typically normalize inputs across all samples. Different inputs can be used, but samples may require different reagent volumes and cycle numbers in later steps based on input.
In a microcentrifuge tube on ice, prepare Hybridize Probe Master Mix with the following volumes per sample (overage included):
Pipette Hybridize Probe Master Mix to mix.
Add 4 uL Master Mix to each well and pipette 10 times to mix.
Seal and place on thermocycler. Select CHMI, Stranded Lig, and run the "HYB DP1" program (95°C for 2 minutes, decrease 0.1°C/sec until 37°C). Keep on the thermocycler until the sample reaches 37°C. Total time is about 15 minutes.
Proceed to rRNA depletion.
STRANDED TOTAL RNA WITH RIBO-ZERO PLUS MICROBIOME: Step 1: Hybridize Probes
Dilute 25-500ng (typically 100ng) total RNA in nuclease-free water to a total volume of 10 uL. Do this in a 96-well PCR plate (if only prepping a few samples, the plate can be cut, but smaller than a half plate can be unstable on the magnet during cleanup steps). We typically normalize inputs across all samples. Different inputs can be used, but samples may require different reagent volumes and cycle numbers in later steps based on input.
In a microcentrifuge tube on ice, prepare Hybridize Probe Master Mix with the following volumes per sample (overage included):
| Name | Volume (µL) |
|---|---|
DB1 (blue cap) | 3.6 µL |
DP1 (blue cap) | 1.2 µl |
DPM (clear cap) | 1.2 µL |
Pipette Hybridize Probe Master Mix to mix.
Add 5 uL Master Mix to each well and pipette 10 times to mix.
Seal and place on thermocycler. Select CHMI, Stranded Lig, and run the "HYB DP1" program (95°C for 2 minutes, decrease 0.1°C/sec until 37°C). Keep on the thermocycler until the sample reaches 37C. Total time is about 15 minutes.
Proceed to rRNA depletion.
In a microcentrifuge tube on ice, prepare rRNA Depletion Master Mix with the following volumes per sample (overage included):
Pipette rRNA Depletion Master Mix to mix.
Centrifuge sealed plate at 280 x g for 10 seconds.
Add 5 uL rRNA Depletion Master Mix to each well, pipette 10 times to mix.
Seal and place on thermocycler and run the "RNA DEP" program (37°C for 15min, 4°C hold).
In a microcentrifuge tube on ice, prepare Probe Removal Master Mix with the following volumes per sample (overage included):
Pipette Master Mix to mix.
Centrifuge sealed plate at 280 x g for 10 seconds.
Add 10 uL Probe Removal Master Mix to each well, pipette 10 times to mix.
Seal and place on thermal cycler and run the program “PRB REM” (37°C for 15min, 70°C for 15min, 4°C hold).
While samples are on the thermal cycler, prepare fresh 80% ethanol if you have not already. If doing the entire protocol in one day, you will need 1.6mL 80% ethanol per sample for the day. Also make sure you have RNAClean XP or AMPure XP beads out at RT.
| NAME | LOCATION | ACTION |
|---|---|---|
Elute, Prime, Fragment High Mix (EPH3) | -20°C Freezer (cDNA synthesis box) | Thaw at RT, vortex and centrifuge briefly |
First Strand Synthesis Act D Mix (FSA) | -20°C Freezer (cDNA synthesis box, brown tube) | Thaw and keep on ice, vortex briefly before use |
This program is about 30 minutes and is your first break.
Step 2: Clean Up and Fragment RNA
Use RNAClean XP or AMPure XP beads for this clean-up. Do not use our homemade beads, which are specific for DNA.
Centrifuge sealed plate at 280 x g for 10 seconds.
Add 60uL well mixed RNAClean XP or AMPure XP beads to each well and pipette to mix thoroughly (or seal plate and vortex for 1 min. Check that the beads are not stuck to the sides of the wells. You may need to spin down the plate and vortex again). Incubate RT for 5 min.
Place on magnet and wait 5 minutes.
Remove and discard 80 uL supernatant.
Keeping plate on the magnetic stand, add 175 uL fresh 80% EtOH to each well. Wait 30 seconds then remove and discard all supernatant.
Repeat EtOH wash.
Remove and discard supernatant. With a p20 pipette, remove and discard any residual supernatant.
Keep on the magnet for 2 minutes to dry.
Remove from the magnet and add 10.5 uL ELB to each well, pipetting to mix until beads are fully resuspended.
Incubate at RT for two minutes
Seal and centrifuge at 280 x g for 10 seconds.
Remove seal, place on magnetic stand, and wait 2 minutes.
Transfer 8.5 uL supernatant from each well to a new plate.
Add 8.5 uL EPH3 to each well, pipette to mix 10 times.
Seal and place on thermocycler and run the "DEN RNA" program.
Generate cDNA
Step 3: First strand cDNA synthesis
| NAME | LOCATION | ACTION |
|---|---|---|
Ampure XP Beads/Homemade Beads | 4°C Fridge | Let stand at RT for 30 minutes to bring to RT, vortex and invert thoroughly before use |
Resuspension Buffer (RSB) | -20°C Freezer (cDNA synthesis box) | Thaw at RT, vortex briefly |
Reverse Transcriptase (RVT) | -20°C Freezer (cDNA synthesis box) | Store until needed. Flick to mix, centrifuge briefly |
Second Strand Marking Mix (SMM) | -20°C Freezer (cDNA synthesis box) | Thaw on ice, invert to mix, centrifuge briefly |
On ice, in a microcentrifuge tube, prepare First Strand Synthesis Master Mix with the following volumes per sample (overage already included):
Pipette First Strand Synthesis Master Mix to mix.
Centrifuge the plate at 280 x g for 10 seconds. Add 8 uL First Strand Synthesis Master Mix to each well and pipette 10 times to mix.
Seal the plate and place in the thermocycler and run the “FSS” program (25°C for 10 min, 42°C for 15 min, 70°C for 15 min, hold at 4°C). Return RVT to the -20°C freezer immediately after use.
This step takes about 40 minutes and is your first large break.
Step 4: Second strand cDNA synthesis
When the first strand synthesis is completed, immediately proceed to the second strand synthesis.
Centrifuge sealed plate at 280 x g for 10 seconds. Add 25 uL SMM to each well. Pipette up and down 10 times.
Seal plate, centrifuge if needed, place plate in thermocycler and run the "SSS" program (16°C for 1hr, hold at 4°C).
Make sure you have Ampure XP or homemade beads at room temp and fresh 80% ethanol for the next steps.
This step takes about 1 hour and is your second large break of the day.
Step 5: cDNA clean-up
When the reaction is complete, add 90 ul of well-mixed AMPure XP or homemade beads to each well containing cDNA and pipette to mix. Incubate RT for 15 min.
Place the plate on the magnet and let sit for 5 min.
Remove and discard 130 ul supernatant per well, being careful not to disturb the beads.
Wash beads on magnet by adding 175 ul of 80% EtOH to each well, without disturbing the beads incubate 30 sec at RT.
Remove and discard the supernatant, repeat the wash step and remove the supernatant again.
Remove any residual supernatant with a p20 pipette. Let the plate stand on the magnet at RT for 2 minutes.
Remove from the magnet and add 19.5 ul of RSB to each well and pipette to fully resuspend the beads. Incubate for 2 min at RT.
Centrifuge at 280 x g for 10 seconds.
Place the plate on the magnetic stand for 2 minutes.
Transfer 17.5 ul of the supernatant (contains the ds cDNA) to new a new plate.
This is a safe stopping point. You can seal the plate and store at -20°C for up to 7 days.
Prepare and Amplify Libraries
Step 6: Adenylate cDNA ends
| NAME | LOCATION | ACTION |
|---|---|---|
RNA Anchor Plate | -20°C Freezer | Thaw at RT, vortex, centrifuge briefly |
DNA/RNA UDI Index Adapter Plate | -20°C Freezer | Thaw at RT, vortex, centrifuge for 1 minute |
A-Tailing Mix (ATL4) | -20°C Freezer (Ligation box) | Thaw at RT, flick to mix, centrifuge briefly |
Stop Ligation Buffer (STL) | -20°C Freezer (Ligation box, red cap) | Thaw at RT, vortex, centrifuge briefly |
Ampure XP Beads/Homemade beads | 4°C Fridge | Let stand at RT for 30 minutes to bring to RT, vortex and invert thoroughly before use |
Resuspension Buffer (RSB) | -20°C Freezer (Ligation box) | Thaw at RT, vortex briefly |
Enhanced PCR Mix (EPM) | -20°C Freezer (Ligation box) | Thaw at RT, invert to mix, centrifuge briefly |
Ligation Mix (LIGX) | -20° Freezer (Ligation box) | Store until needed, flick to mix, centrifuge briefly |
Add 12.5 ul of thawed A-tailing mix to each well. Using a p200 pipette set to 30 µL, pipette up and down 10 times.
Seal and put on the thermocycler for the "ATAIL" program (37°C for 30min, 70°C for 5min, 4°C hold).
This step takes about 35 minutes and is another break.
Step 7: Ligate Anchors
Each well of the anchor plate is single-use and contains the same RNA Index Anchors. They can be used in any order. Seal the used anchor wells with foil and label before returning to freezer storage.
| Order of Addition | Reagent | Vol for Sample Input < or = 100ng | Vol for Sample Input > 100ng |
|---|---|---|---|
1 | RSB | 2.5 uL | 0 uL |
2 | RNA Index Anchors | 2.5 uL | 5 uL |
3 | LIGX | 2.5 uL | 2.5 uL |
With a p200 pipette set to 35 µL, pipette to mix 10 times.
Seal and run on the thermocycler under the "LIG" program (30°C for 10min, 4°C hold).
Briefly centrifuge plate. Add 5 uL STL to each well, pipetting to mix 15 times.
Step 8: Clean Up Adapter-Ligated Fragments
Add 34 ul of AMPure XP or homemade beads to each reaction, mix well by pipetting, and incubate at RT for 15 min.
Place on magnetic stand for 5 min at RT.
With the plate on the magnetic stand, remove 67 ul of supernatant from each sample, being careful not to disturb the beads.
With the plate on the magnetic stand, wash with 175 ul 80% EtOH for 30 sec, then remove the ethanol.
Perform the wash step again.
Use a p20 pipette to remove residual ethanol. Keep the plate on the magnet at RT for 2 minutes to dry.
Remove the plate from the magnetic stand and add 22 ul of Resuspension Buffer to each well, and mix well by pipetting to fully resuspend beads.
Incubate at RT for 2 minutes.
Centrifuge at 280 x g for 10 seconds.
Place the plate on magnet for 5 min.
Keeping the plate on the magnet, recover 20 ul from each well and transfer to a new plate.
This is a safe stopping point. You can seal the plate and store at -20°C for up to 7 days.
Step 9: Index and Amplify library
Add 10 uL index adapter to each well.
Indexes come in a 96 well plate, each well containing a unique set of dual indexes. Although they are unique, certain patterns of index use are recommended to balance color channels during sequencing. See Illumina's Index Adapters Pooling Guide. Generally, moving in columns is fine. Seal the used wells with foil and label before returning to freezer storage. Record which index well was used for each sample, as this is needed to demultiplex after sequencing.
Add 20 uL EPM. Pipette 10 times to mix.
Seal and place on thermocycler and run the "PCR" program, with cycle repeats according to the table below.
This is a 30-45 minute break, depending on cycle number
Step 10: Clean Up Library
Add 50 ul of AMPure or homemade beads to each well and mix well by pipetting. Incubate 15 min at RT.
Place on the magnet and incubate for 5 minutes.
Remove and discard 90 ul of supernatant from each well.
Wash the beads 175 ul/well of 80% EtOH, wait 30s, remove the supernatant.
Repeat the ethanol wash step.
Use a p20 pipette to remove residual ethanol.
Keep the plate on the magnet at RT for 2 minutes to dry.
Remove the plate from the magnetic stand, add 17 ul of resuspension buffer and mix well by pipetting.
Incubate for 2 min at room temperature.
Place the plate back on the magnetic stand for 5 minutes.
Remove 15 uL of supernatant from each sample and move it into a new plate. This is the final cDNA product.
Congratulations! You’re done!…well, almost
Quality Checks
Perform quality control steps:
Tapestation to determine the library size
Qubit to quantify the libraries
Libraries can be stored at -20 for up to 30 days.
Sequencing Guidelines
Normalize and Pool
Quantify each of your libraries on Qubit. For most libraries, using the HS dsDNA Qubit assay with 2uL of input will yield a reading. Record the concentration in ng/uL for each library.
Run your samples on Tapestation with either the D1000 or the HSD1000 assay. Remember to allow Tapestation reagents to sit at room temperature for at least 30 minutes before use. Save your Tapestation results by going to File -> Create Report -> Save as pdf. This file can then be emailed or uploaded to Asana. For the base pair length, we usually use the value of the peak identified by the Tapestation analysis software. This value is shown both on the tracing itself and in the Peak Table for each sample.
Download our nM Conversion Calculator here. Enter the concentration (from Qubit) and the base pair length (from Tapestation) in the appropriate cells and it will give you the nM concentration for each library. Normalize and pool all your libraries 2 nM in a LoBind microcentrifuge tube. If you need to dilute your libraries, we recommend using at least 2uL to minimize pipetting errors. The example sheet of the calculator provides further detail.
Quantify your pool on Qubit and enter into the calculator sheet to check that your pool is close to the nM concentration you normalized to.