- Materials
-
- 4 ng enriched RNA (Poly(A)+ RNA or ribodepleted) or 200 ng total RNA
- cDNA RT Adapter (CRTA)
- Annealing Buffer (AB)
- Short Fragment Buffer (SFB)
- RT Primer (RTP)
- Strand Switching Primer II (SSPII)
- Consumables
-
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- NEBNext® Quick Ligation Reaction Buffer (NEB, B6058)
- T4 DNA Ligase 2M U/ml (NEB, cat # M0202M)
- Lambda Exonuclease (NEB, Cat # M0262L)
- USER (Uracil-Specific Excision Reagent) Enzyme (NEB, cat # M5505L)
- Agencourt RNAClean XP beads (Beckman Coulter™, cat # A63987)
- 10 mM dNTP solution (e.g. NEB cat # N0447)
- Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0751)
- RNaseOUT™, 40 U/μl (Life Technologies, cat # 10777019)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Qubit RNA HS Assay Kit (ThermoFisher, cat # Q32852)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
-
- Microfuge
- Thermal cycler
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Qubit fluorometer (or equivalent for QC check)
-
Thaw the following reagents, then spin down briefly using a microfuge and mix as indicated in the table below. Then place the reagents on ice.
Reagent 1. Thaw at room temperature 2. Briefly spin down 3. Mix well by pipetting cDNA RT Adapter (CRTA) ✓ ✓ ✓ Annealing Buffer (AB) ✓ ✓ ✓ Short Fragment Buffer (SFB) ✓ ✓ ✓ RT Primer (RTP) ✓ ✓ ✓ Strand Switching Primer II (SSPII) ✓ ✓ ✓ NEBNext® Quick Ligation Reaction Buffer ✓ ✓ Mix by vortexing T4 DNA Ligase 2M U/ml Not frozen ✓ ✓ RNaseOUT Not frozen ✓ ✓ Lambda Exonuclease Not frozen ✓ ✓ Uracil-Specific Excision Reagent (USER) Not frozen ✓ ✓ 10 mM dNTP solution ✓ ✓ ✓ Maxima H Minus Reverse Transcriptase Not frozen ✓ ✓ Maxima H Minus 5x RT Buffer ✓ ✓ Mix by vortexing -
Optional actionTo run a control experiment, replace your sample input with 10 μl diluted RNA Control Sample (RCS) from the RNA Control Expansion (EXP-RCS001) as follows:
- Thaw the RNA Control Sample (RCS) at room temperature, briefly spin down and mix well by pipetting.
- Dilute the RNA Control Sample (RCS) in a 1.5 ml Eppendorf DNA LoBind tube as follows:
Reagent Volume RNA Control Sample (RCS) 1 μl Nuclease-free water 14 μl Total 15 μl Note: This will provide enough volume for 3 samples, adjust your volumes accordingly for the number of samples you wish to run in your control experiment.
- Mix thoroughly by pipetting 10-20 times and briefly spin down.
- Take forward 4 μl of the diluted RNA Control Sample (RCS) per sample and make up each volume to 10 μl with nuclease-free water as follows:
Reagent Volume Diluted RNA Control Sample (RCS) 4 μl Nuclease-free water 6 μl Total volume per sample 10 μl - Mix by flicking the tube and spin down.
- Use the 10 μl of diluted RNA Control Sample (RCS) as your RNA input.
-
For each sample, prepare the RNA in nuclease-free water.
- Transfer 4 ng Poly(A)+ RNA, or 200 ng total RNA into a 1.5 ml Eppendorf DNA LoBind tube
- Adjust the volume up to 10 µl with nuclease-free water
- Mix by flicking the tube to avoid unwanted shearing
- Spin down briefly in a microfuge
-
Prepare the following in a 0.2 ml PCR tube per sample:
Reagent Volume RNA 10 µl cDNA RT Adapter (CRTA) 1 µl Annealing Buffer (AB) 1 µl Total volume 12 µl -
Mix gently by flicking the tubes, and spin down.
-
Incubate the reactions in the thermal cycler at 60°C for 5 mins, then cool for 10 minutes at room temperature.
-
To each of the 0.2 ml PCR tubes containing you RNA sample(s), add the following:
Reaction Volume RNA sample (from previous step) 12 µl NEBNext® Quick Ligation Reaction Buffer 3.6 µl T4 DNA Ligase 2M U/ml 1.4 µl RNaseOUT 1 µl Total volume (including all reagents) 18 µl -
Ensure the components are thoroughly mixed by flicking the tubes and spin down.
-
Incubate for 10 minutes at room temperature.
-
To each of the 0.2 ml PCR tubes, add the following:
Reagent Volume RNA sample (from previous step) 18 µl Lambda Exonuclease 1 µl USER (Uracil-Specific Excision Reagent) 1 µl Total volume (including all reagents) 20 µl -
Ensure the components are thoroughly mixed by flicking the tubes and spin down.
-
Incubate for 15 minutes at 37°C in the thermal cycler.
-
Transfer each sample to clean 1.5 ml Eppendorf DNA LoBind tubes.
-
Resuspend the RNase-free XP beads by vortexing.
-
Add 36 µl of resuspended RNase-free XP beads to each reaction and mix gently by flicking the tubes.
-
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
-
Spin down the samples and pellet on a magnet. Keep the tubes on the magnet, and pipette off the supernatant.
-
Keep the tubes on the magnet and wash the beads with 100 µl of Short Fragment Buffer (SFB) without disturbing the pellet. Remove the SFB using a pipette and discard.
-
Repeat the previous step.
-
Spin down and place the tubes back on the magnet. Pipette off any residual buffer. Briefly allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
-
Remove the tubes from the magnetic rack and resuspend each pellet in 12 µl of nuclease-free water.
-
Incubate at room temperature for 10 minutes.
-
Pellet the beads on a magnet until the eluate is clear and colourless.
-
Remove and retain 12 µl of eluate into a clean 0.2 ml thin-walled PCR tube per sample.
-
To each of the 0.2 ml PCR tubes, add the following:
Reagents Volume Eluted sample (from previous step) 12 µl RT Primer (RTP) 1 µl dNTPs (10 mM) 1 µl Total volume (including all reagents) 14 µl -
Ensure the components are thoroughly mixed by flicking the tubes and spin down.
-
Incubate the reaction for 15 minutes at room temperature.
-
To each of the 0.2 ml PCR tubes, add the following:
Reagents Volume RT primed sample (from previous step) 14 µl Maxima H Minus 5x RT Buffer 4.5 µl RNaseOUT 1 µl Strand Switching Primer II (SSPII) 2 µl Total (including all reagents) 21.5 µl -
Mix gently by flicking the tubes, and spin down.
-
Incubate at 42°C for 2 minutes in the thermal cycler.
-
Add 1 µl of Maxima H Minus Reverse Transcriptase to each tube. The total volume will be 22.5 µl per tube.
-
Mix gently by flicking the tubes, and spin down.
-
Incubate using the following protocol using a thermal cycler:
Cycle step Temperature Time No. of cycles Reverse transcription and strand-switching 42°C 90 mins 1 Heat inactivation 85°C 5 mins 1 Hold 4°C ∞