(A) RNA Samples:
- Prepare 4-5 μg of total RNA for the protocol.
- Ensure high quality total RNA is obtained, either through preparation or purchase from commercial sources.
- Test the integrity of the RNA samples by evaluating the 28S / 18S rRNA ratio using a Bioanalyzer or Tapestation (Agilent Technologies).
- Aim for RNA integrity number (RIN) values of ≥8.5 for optimal results. Process samples with lower RIN values if necessary, but be prepared for potential replication to reach the desired cDNA amount for downstream applications.
- Check sample purity by measuring the ratio of absorbance at 260 / 280 nm using a Nanodrop spectrophotometer (Nanodrop One full-spectrum - Thermo Scientific).
- Quantify RNA samples using UV absorbance–based quantification with a Qubit fluorometer (Thermo Fisher Scientific) and Qubit RNA assay kit.
(B) Adding Exogenous Reference Transcripts (Spike-ins):
Utilize exogenous synthetic references such as Lexogen SIRVs and ERCC spike-ins to monitor sample performance and reproducibility. Note that spike-in molecules are typically provided in an uncapped form by manufacturers. Perform artificial 5’ capping on spike-in mixes before use, for example, using the Vaccinia system, to prevent complete loss during cap selection steps.
- Dilute 1 μl of 5’ capped synthetic controls (SIRVs and ERCC) at a ratio of 1:100.
- Add the diluted synthetic controls to 5 μg of the RNA sample prior to initiating the CapTrap protocol.
- Adjust the dilution according to the desired concentration and the mass of the RNA sample and spike-ins.
- Expect approximately 0.80% of sequencing reads to originate from spike-in sequences based on the applied dilution and mass of the sample and spike-ins.
(C) First-strand Synthesis and Purification:
- Prepare the hybridization of oligo dT primer mix. Mix together the specified amounts of total RNA (5 μg) and Spike-in Mix, 1 μl dNTPs (10 mM), and 0.5 μl oligo dT16VN primer. Adjust the volume with UltraPure DNase/RNase-Free Distilled Water to reach a total volume of 10 μl.
- Incubate the mixture at 65ºC for 5 minutes, followed by cooling on ice for 1 minute.
- Prepare the enzyme mix and keep it on ice until needed. Combine 4 μl of PrimeScript II Buffer (5x), 1 μl RNasin Plus RNase inhibitor, 4 μl UltraPure DNase/RNase-Free Distilled Water, and 1 μl PrimeScript II enzyme. Ensure the total volume reaches 10 μl.
- Add 10 μl of the enzyme mix to each RNA/primer mix reaction, making a total volume of 20 μl per reaction.
- Gently mix the reaction and quickly spin the samples.
- Incubate the reaction at 42ºC for 60 minutes and hold the samples at 4ºC.
- After the first-strand synthesis incubation step, purify the sample using 1.8x AMPure RNA Clean XP beads.
- Incubate the sample with the beads for 5 minutes at room temperature (RT), then place the sample on a magnet and wait for the solution to clear.
- Remove the supernatant and wash the beads twice with freshly prepared 70% ethanol solution without disturbing the beads.
- Air dry the beads for 1 minute (avoid beads overdrying), then resuspend the sample in 42 μl of nuclease-free water.
- Incubate the sample at 37ºC for 5 minutes in a thermo block with gentle rotation.
- Place the sample on the magnet and wait for the solution to clear, then recover 40 μl of supernatant and transfer it into a clean tube.
OPTIONAL STOPPING POINT. First-strand hybrid ssRNA + sscDNA can be stored for 1 hour on ice or at -20ºC overnight as a stopping point.
(D) Cap Selection:
1. Oxidation
1.1 Combine 40 µl of the sample from the previous step with 2 µl of NaOAc (1M, pH 4.5) and 2 µl of NaIO4 (250 mM).
1.2 Mix well by pipetting and wrap the samples with aluminum foil.
1.3 Incubate on ice for 5 minutes.
1.4 Stop the oxidation by adding 16 µl of Tris HCl (1M pH 8.5) and mix well by pipetting.
1.5 Purify the sample using 1.8x AMPure RNA Clean XP beads.
2. Biotinylation
2.1 Combine 40 µl of the sample from the previous step with 4 µl of NaOAc (1M, pH 6) and 4 µl of Biotin (long arm Hydrazide) (100 mM).
2.2 Mix well by pipetting and spin down the sample.
2.3 Incubate at 40ºC for 30 minutes.
2.4 Purify the sample using 1.8x AMPure RNA Clean XP beads.
3. RNase ONE Treatment and Purification
3.1 Combine 40 µl of the sample from the previous step with 4.5 µl of RNase ONE buffer (10x) and 0.5 µl of RNase ONE (10 U/µl).
3.2 Mix well by pipetting and spin down the sample.
3.3 Incubate at 37ºC for 30 minutes.
3.4 Purify the sample using 1.8x AMPure RNA Clean XP beads.
OPTIONAL STOPPING POINT. After RNAse ONE treatment and Purification, the first strand hybrid ssRNA + sscDNA can be stored for 1 hour on ice or at -20ºC overnight.
4. Cap-trapping
4.1 Recondition Magnetic streptavidin beads (M-270) with LiCl binding buffer: 30 µl of Magnetic streptavidin beads (M-270) for each sample and use the LiCl binding buffer to recondition the beads for Cap-trapping step. Scale up volume for multiple samples. Stand the tube containing the beads at the magnetic rack for 2-3 minutes and remove the supernatant. Wash the beads 2 times with 1 volume (30 µl for 1 sample) of LiCl binding buffer. Vortex the beads and spin between the 2 washes. Resuspend the beads with 95 µl of LiCl binding buffer.
4.2 Combine 40 µl of the sample from the previous step with 95 µl of Magnetic beads with LiCl binding buffer. 4.3 Mix gently by pipetting and incubate at 37ºC for 15 minutes.
4.4 Wash the beads with 150 µl of TE wash buffer containing 10% Tween 20.
5. First-strand Release, Purification, and Concentration
5.1 Prepare M-270 beads for the first release and heat shock at 95ºC for 5 minutes.
5.2 Collect the supernatant in a new tube and keep it on ice.
5.3 Prepare M-270 beads for the second release.
5.4 Mix the second-release supernatant with the first-release supernatant.
5.5 Prepare a mix containing 0.10 µl of RNase H (60 U/µl), 2 µl of RNase ONE (10 U/µl), and 2.90 µl of Release buffer per sample.
5.6 Add 5 µl of this mix to each sample.
5.7 Incubate at 37ºC for 30 minutes.
5.8 Purify the sample using 1.8x AMPure DNA XP beads.
OPTIONAL STOPPING POINT. First strand sscDNA can be stored for 1 hour on ice or at -20ºC overnight.
(E) Cap-dependent Linker Ligation:
1. 5’ Linker Ligation, Purification, and Concentration
1.1 Dry 40 μl of the sample from the previous step using a speed vac concentrator at 80ºC for 35 minutes.
1.2 After drying, add 4 μl of nuclease-free water and carefully resuspend the lyophilized pellet.
1.3 Incubate the 4 μl of sample at 95ºC for 5 minutes and quickly place it on ice for 2 minutes. Briefly spin down the tube containing the sample.
1.4 Denature 1 μl of CapTrap 5’ linker for each sample at 55 ºC for 5 minutes and quickly place it on ice for 2 minutes. Briefly spin down the tube containing the linker.
1.5 Prepare the 5’ linker ligation mix by combining 4 μl of sample, 1 μl of CapTrap 5’ linker, and 10 μl of Mighty mix.
1.6 Incubate at 37 ºC for 4 hours or at 16 ºC overnight.
1.7 Add 35 μl of nuclease-free water to the sample and purify the resulting 50 µl using 1.8x AMPure DNA XP beads.
1.8 Perform a second purification step by adding 1.8x AMPure DNA XP beads to the 40 μl of sample from the first purification.
2. 3’ Linker Ligation, Purification, and Concentration
2.1 Take 40 μl of the sample resulting from the 5’ linker ligation step and dry them using a speed vac concentrator at 80ºC for 35 minutes.
2.2 After drying, add 4 μl of nuclease-free water and carefully resuspend the lyophilized pellet.
2.3 Incubate the 4 μl of sample at 95ºC for 5 minutes and quickly place it on ice for 2 minutes. Briefly spin down the tube containing the sample.
2.4 Denature 1 μl of CapTrap 3’ linker for each sample at 65 ºC for 5 minutes and quickly place it on ice for 2 minutes. Briefly spin down the tube containing the linker.
2.5 Prepare the 3’ linker ligation mix by combining 4 μl of the sample, 1 μl of CapTrap 3’ linker, and 10 μl of Mighty mix.
2.6 Incubate at 37 ºC for 4 hours or at 16 ºC overnight.
2.7 Add 35 μl of nuclease-free water to the sample and purify the resulting 50 µl using 1.8x AMPure DNA XP beads.
2.8 Perform a second purification step by adding 1.8x AMPure DNA XP beads to the 40 μl of sample from the first purification.
3. SAP & USER Treatment
3.1 Prepare the enzyme mix by combining 40 μl of the sample from the previous step with 2 μl of UltraPure DNase/RNase-Free Distilled Water, 5 μl of SAP Buffer (10x), 1 μl of SAP enzyme (1 U/μl), and 2 μl of USER enzyme (1 U/μl).
3.2 Incubate at 37ºC for 30 minutes followed by a step at 95ºC for 5 minutes and quickly place everything on ice.
3.3 Purify the resulting 50 µl using 1.8x AMPure DNA XP beads.
3.4 Perform a second purification step by adding 1.8x AMPure DNA XP beads to the 40 μl of sample from the first purification.
OPTIONAL STOPPING POINT. First strand sscDNA can be stored for 1 hour on ice or at -20ºC overnight.
(F) Full-length cDNA Library Synthesis:
1. Second Strand Synthesis, Purification, and Concentration
1.1 Dry the 40 μl of sample from the USER & SAP treatment step using a speed vac concentrator at 80ºC for 35 minutes.
1.2 After drying, add 5 μl of nuclease-free water and carefully resuspend the lyophilized pellet.
1.3 Prepare the second strand synthesis mix by combining 5 μl of sample, 12.5 μl of HiFi KAPA mix 2x, 0.5 μl of 2nd strand UMI primer, 1.3 μl of DMSO, and 5.8 μl of UltraPure DNase/RNase-Free Distilled Water.
1.4 Incubate the mixture for 5 minutes at 95ºC, 5 minutes at 55ºC, 30 minutes at 72ºC, and finally hold at 4ºC.
1.5 Treat the second strand synthesis reaction by adding 1 µl of Exonuclease I (20 U/µl). Mix carefully and quickly spin.
1.6 Incubate the mixture for 30 minutes at 37 ºC. 1.7 Purify the sample by adding 1.8x AMPure DNA XP beads and perform a second purification step by adding 1.4x AMPure DNA XP beads.
2. cDNA Library Enrichment (LA PCR)
2.1 Dry up the samples with speed vac (75 minutes at 37ºC) and carefully resuspend them with 5 μl of nuclease-free water.
2.2 Split the 5 μl of each sample into two PCR independent reactions.
2.3 Assemble each PCR reaction mix by combining 2.5 μl of sample, 29 μl of UltraPure DNase/RNase-Free Distilled Water, 5 μl of 10x Buffer, 8 μl of dNTPs, 2.5 μl of Primer FOR, 2.5 μl of Primer REV, and 0.5 μl of Taq LA Takara.
2.4 Use the following PCR cycling conditions: 30 seconds at 95°C for initial denaturation, followed by 16 cycles of 15 seconds at 95°C, 15 seconds at 55°C, and 8 minutes at 65°C for amplification. After the cycles, extend for 10 minutes at 65°C and then hold at 4°C. Perform 10-20 PCR cycles depending on the amount of required full-length cDNA for downstream applications.
2.5 Merge the 2 PCR replicates and purify with 1x AMPure DNA XP beads.
3. Quantification and Quality Check
3.1 Verify cDNA amount and concentration using a Qubit quantitation platform.
3.2 Use the other 1 μL of sample for cDNA sizing QC in Bioanalyzer.
4. Storage
CapTrap cDNA enriched libraries can be stored at -20ºC until sequencing library preparation or other downstream applications.
(G) ONT Template Preparation:
1. Sample Repair and A-Tailing of CapTrap cDNA Libraries
1.1 Normalize 500 ng of CapTrap cDNA enriched libraries to a final volume of 47 µl using UltraPure DNase/RNase-Free Distilled Water.
1.2 Prepare the reaction mixture by combining CapTrap cDNA, 1 µl DNA CS (DCS), 3.5 µl NEBNext FFPE DNA Repair Buffer, 2 µl NEBNext FFPE DNA Repair Mix, 3.5 µl Ultra II End-prep reaction Buffer, and 3 µl Ultra II End-prep enzyme mix.
1.3 Incubate the mixture at +20 °C for 5 minutes, then at +65 °C for 5 minutes, followed by a quick spin.
1.4 Purify the sample using AMPure DNA XP beads, elute it in 60 µl nuclease-free water, and transfer it to a clean tube.
2. Adapter Ligation
2.1 Prepare the ligation reaction by combining the sample from the previous step 60 µl with 25 µl Ligation Buffer (LNB), 10 µl NEBNext Quick T4 DNA ligase, and 5 µl Adapter Mix (AMX).
2.2 Incubate the mixture at RT for 10 minutes, then purify the sample using AMPure DNA XP beads and elute it in 15 µl Elution Buffer (ELB).
2.3 Quantify the eluted sample using a Qubit fluorometer and store it on ice until ready to load.
3. Priming the SpotON Flow Cell
3.1 Prepare the flow cell priming mix by combining the Flush Buffer (FB) vial and 30 µl Flush Tether (FLT).
3.2 Load 800 µl of Priming mix into the flow cell via the priming port, then incubate at RT for 5 minutes.
4. Library Loading
4.1 Prepare the library loading mix by combining 37.5 µl of Sequencing Buffer (SQB), 25.5 µl of Library Loading Beads (LB), and 150 fmol of cDNA MinION library (adjusted to the final volume using ELB). Adjust the volume to 12 µl using ELB.
4.2 Complete the flow cell priming by loading 200 µl of the priming mix into the flow cell via the priming port with the SpotON port open.
4.3 Gently mix the prepared library loading mix and load 75 µl into the flow cell via the SpotON port drop by drop.
4.4 Close priming and SpotON ports and start the run.
(H) PacBio Template Preparation:
1. cDNA Damage Repair
1.1 Normalize 500 ng of CapTrap cDNA enriched libraries to a final volume of 47 µl using UltraPure DNase/RNase-Free Distilled Water.
1.2 For each sample, add the following components in the order listed:
- UltraPure DNase/RNase-Free Distilled Water: x µl
- CapTrap cDNA from previous step (500 ng): x µl
- DNA prep buffer: 7 µl
- NAD: 0.6 µl
- DNA Damage Repair Mix: 2 µl
1.3 Pipette mix 10 times without flicking the tube. Spin down the contents with a quick spin and incubate at 37°C for 30 minutes. Return the reaction to 4°C.
2. Repair Ends/A-Tailing
2.1 For each sample, add the following components:
- Sample from the previous step: 57 µl
- End Prep Mix: 3 µl
2.2 Pipette mix 10 times without flicking the tube. Spin down the contents and incubate at 20°C for 10 minutes followed by 65°C for 30 minutes. Return the reaction to 4°C.
3. Adapter Ligation
3.1 For each sample, add the following components:
- A-tail sample from the previous step: 60 µl
- Adapter: 3 µl
3.2 Mix well by pipetting 10 times. Then add:
- Ligation Mix: 30 µl
- Ligation enhancer: 1 µl
- Ligation additive: 1 µl
3.3 Pipette mix 10 times without flicking the tube. Spin down the contents and incubate at 20°C for 60 minutes. Return the reaction to 4°C.
4. Purify cDNA SMRTbell Templates
Purify the sample by adding 1x Pronex beads to the 95 µl reaction. Mix by pipetting 15 times without flicking the tube. Quick spin and incubate at room temperature for 5 minutes. Place the sample on the magnet, remove the supernatant, and wash twice with 80% ethanol solution. Air dry for 1 minute, then resuspend in 12 µl of elution buffer. Incubate at room temperature for 5 minutes, place on the magnet, and recover 11 µl of supernatant.
5. Quality Check and Storage
Verify cDNA amount and concentration using a Qubit quantitation platform. Perform cDNA sizing QC using Bioanalyzer. Store CapTrap SMRTbell sequencing libraries at -20ºC until ready for sequencing.