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- Documentation
- A few important comments before you start
- Purify and Fragment mRNA
- Step 1: Capture mRNA
- Step 2: Clean Up and Fragment mRNA
- 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
- Sequencing Guidelines
- Normalize and Pool
- Setup Run in Basespace
- Loading the Sequencer
Documentation
Illumina's Stranded mRNA Sample Prep Guide. This method makes a cDNA library of the polyadenylated mRNA in your eukaryotic samples of interest. It requires high quality RNA with intact polyA tails.
A few important comments before you start
- The recommended starting amount of total RNA is 25-1000ng, but we usually try to stay away from the extreme ends of this spectrum and use 100ng input.
- 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 hours if completing the protocol in one day. This estimate is based on an experienced user with a multichannel pipette and vortexing at bead cleanup steps. An inexperienced user or one not using time-saving techniques should plan for more time.
- Keep all reagents on ice unless otherwise stated. Do NOT put any reagents stored at 4C, 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 the resuspension buffer and the 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 vortexing 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.
Purify and Fragment mRNA
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 1 box of reagents in the 4C fridge and 2 boxes of reagents in the -20C freezer, each labeled with a green dot saying "mRNA Ligation". 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 |
---|---|---|
Bead Binding Buffer (BBB) | 4C Fridge | Vortex briefly, leave at RT |
Bead Washing Buffer (BWB) | 4C Fridge | Vortex briefly, leave at RT |
Elution Buffer (EB) | 4C Fridge | Vortex briefly, leave at RT |
RNA Purification Beads (RPBX) | 4C Fridge | Let stand at RT for 30 minutes to bring to RT, vortex and invert thoroughly before use |
Elute, Prime, Fragment High Mix (EPH3) | -20 Freezer (cDNA synthesis box, gray sticker) | Thaw at RT, vortex briefly |
First Strand Synthesis Act D mix (FSA) | -20 Freezer (cDNA synthesis box, brown tube) | Thaw and keep on ice, vortex briefly before use |
NAME | LOCATION | ACTION | VOLUME |
---|---|---|---|
Ampure XP Beads | 4C Fridge | Let stand at RT for 30 minutes to bring to RT, vortex and invert thoroughly before use | 174uL per sample for entire prep |
Fresh 80% Ethanol (200 proof EtOH + MilliQ water) | RT Bench | Mix fresh each day. Low% EtOH negatively affects cleanups. | 1.2mL per sample for entire prep |
RNAse Free RT-PCR Grade Water | -20C Freezer | Thaw at RT | As needed for diluting RNA input |
Post-Style Magnetic Stand for 96 well plates | RT Bench |
Step 1: Capture mRNA
Do not store RNA 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.
Step 2: Clean Up and Fragment mRNA
Generate cDNA
Step 3: First strand cDNA synthesis
NAME | LOCATION | ACTION |
---|---|---|
Ampure XP Beads | 4C Fridge | Let stand at RT for 30 minutes to bring to RT, vortex and invert thoroughly before use |
Resuspension Buffer (RSB) | -20 Freezer (cDNA synthesis box, purple sticker) | Thaw at RT, vortex briefly |
Reverse Transcriptase (RVT) | -20 Freezer (cDNA synthesis box, pink sticker) | Store until needed. Flick to mix, centrifuge briefly |
Second Strand Marking Mix (SMM) | -20 Freezer (cDNA synthesis box, blue sticker) | Thaw on ice, invert to mix, centrifuge briefly |
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.
This step takes about 1 hour and is your second large break of the day.
Step 5: cDNA clean-up
This is a safe stopping point. You can seal the plate and store at -20C for up to 7 days.
Prepare and Amplify Libraries
Step 6: Adenylate cDNA ends
NAME | LOCATION | ACTION |
---|---|---|
Anchor Plate | -20 Freezer | Thaw at RT, vortex, centrifuge briefly |
Index Adapter Plate | -20 Freezer | Thaw at RT, vortex, centrifuge for 1 minute |
A-Tailing Mix (ATL4) | -20 Freezer (Ligation box, green sticker) | Thaw at RT, flick to mix, centrifuge briefly |
Stop Ligation Buffer (STL) | -20 Freezer (Ligation box, red cap) | Thaw at RT, vortex, centrifuge briefly |
Ampure XP Beads | 4C Fridge | Let stand at RT for 30 minutes to bring to RT, vortex and invert thoroughly before use |
Resuspension Buffer (RSB) | -20 Freezer (cDNA synthesis box, purple sticker) | Thaw at RT, vortex briefly |
Enhanced PCR Mix (EPM) | -20 Freezer (Ligation box, yellow sticker) | Thaw at RT, invert to mix, centrifuge briefly |
Ligation Mix (LIGX) | -20 Freezer (Ligation box, red sticker) | Store until needed, flick to mix, centrifuge briefly |
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 |
Step 8: Clean Up Adapter-Ligated Fragments
This is a safe stopping point. You can seal the plate and store at -20C for up to 7 days.
Step 9: Index and Amplify library
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 plate (Set A or B) and which index well was used for each sample, as this is needed to demultiplex after sequencing.
This is a 30-45 minute break, depending on cycle number
Step 10: Clean Up Library
Congratulations! You’re done!…well, almost
Perform quality control steps - we use Tapestation to determine the library size and Qubit to quantify the libraries. More detail on QC and sequencing is below.
Libraries can be stored at -20 for up to 30 days.
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.
- The recommended loading concentration is 1.6pM and 5% PhiX spike-in.