Poly(A)+ RNA preparation (using Illumina Truseq mRNA preparation kit).
1. Dilute 2-10 μg of total RNA in 50 μl of RNase-free H2O.
2. Vortex RNA Purification Beads and add 50 μl to RNA sample. Pipet up and down 6 times to mix.
3. Incubate in thermocycler at 65˚ C for 5 min., on ice for 5 min. and at room temperature for 5 min.
4. Place sample in magnetic separator for 5 min. Remove and discard all the supernatant.
5. Remove sample from rack. Add 200 μl of Bead Washing Buffer and pipet up and down 6 times to mix sample.
6. Place the sample back in the magnetic separator for 5 min. Remove and discard all the supernatant.
7. Add 50 μl of Elution Buffer and pipet up and down 6 times.
8. Incubate in thermocycler at 80˚ C for 2 min.
9. Remove sample from thermocycler and keep at room temperature for 5 min.
10. Add 50 μl of Bead Binding Buffer and pipet up and down 6 times. Incubate at room temperature for 5 min.
11. Place sample in magnetic separator for 5 min. Remove and discard all supernatant.
12. Remove sample from rack. Add 200 μl of Bead Washing Buffer and pipet up and down 6 times.
13. Place sample in magnetic separator for 5 min. Remove and discard all supernatant.
14. Add 16 μl of RNase-free H2O and pipet up and down 6 times.
15. Incubate in thermocycler at 70˚ C for 2 min., then on ice for 1 min.
16. Place sample in magnetic rack for 5 min. Transfer 16 μl of the supernatant to a new 0.2 ml PCR tube. Keep 1 μl for Bioanalyzer (optional).
GI tailing of RNA (using USB poly(A) tail length assay, Thermo Fisher).
1. Prepare the following mix, keeping the reagents on ice.
poly(A)+ RNA: 14 μl
5X tail buffer mix: 4 μl
10X tail enzyme mix: 2 μl
Tot. vol.: 20 μl
Incubate at 37°C for 60 min.
2. Add 1.5 μl of tail stop solution, keep on ice for 2 min.
3. Add 1.8X XP RNA beads, incubate at room temperature for 5 min.
4. Put on magnetic rack, keep for 3 min. Remove supernatant.
5. Wash twice with 50 μl Ethanol 80% for 30 sec.
6. Carefully remove supernatant, leave tubes open for 10 min in order to dry.
7. Resuspend beads in 17 μl H2O, put on magnetic rack for 5 min. and collect supernatant. Keep 1 μl for Bioanalyzer (optional).
cDNA synthesis (using SMARTscribe reverse transcriptase kit, Clontech).
1. Prepare the following mix:
5X First strand buffer: 8 μl
DTT 20mM: 1.5 µl
dNTP mix 10 mM: 4 µl
RNase Inhibitor: 2 μl
isoTSO 12 µM: 2 µl
SMARTScribe RT: 2 µl
H2O: 2.5 µl
Tot. vol.: 22 µl
Keep the master mix at room temperature.
2. In a second PCR tube, mix 16 µl of purified GI-tailed RNA and 2 µl of RT primer (10µM).
3. Mix contents and spin briefly. Incubate in thermocycler starting the following program: 72°C for 3 min., 42°C for 60 min., 70 °C for 10 min. and at 4°C hold.
4. When the RNA/primer sample has done completed the first step at 72°C, wait for 1-2 min. until it reaches the temperature of 42°C and add the RT master mix, previously equilibrated at room temperature, reaching a final volume 40µl.
5. When the reaction is finished, add 0.6X XP DNA beads, incubate at room temperature for 5 min.
6. Put on magnetic rack, keep for 3 min. Remove supernatant.
7. Wash twice with 50 μl Ethanol 80% for 30 sec.
8. Carefully remove supernatant, leave tubes open for 10 min.
9. Resuspend beads in 42 µl of H2O, put on magnetic rack for 5 min. and collect supernatant. Keep 1 µl for Bioanalyzer (optional).
cDNA library amplification (using Advantage 2 PCR enzyme system, Clontech).
1.Prepare the following mix:
10X Advantage 2SA PCR buffer: 10 μl
Diluted first-strand cDNA: 40 μl
dNTP Mix (10 mM each): 2μl
5’ PCR Primer II A (10 μM): 2 μl
Univ. RV Primer (10 µM): 2 µl
H2O: 42 µl
50X Advantage 2 Polymerase Mix: 2 μl
Tot. vol.: 100 μl
Keep the master mix on ice.
2. Start the thermocycler with the following program: 98°C for 1 min., N. cycles x [98°C for 10 sec., 63°C for 15 sec., 68°C for 3 min.], 68°C for 7 min. Put the tube into the thermocycler when it has reached the temperature of 98°C. Note: if the sample has never been processed before, do PCR optimization splitting the 100 μl reaction into 4 tubes and do 18, 20, 22, 24 cycles, then compare the amplified libraries in terms of size profile and yield.
3. When the reaction is finished, add 0.6X XP DNA beads, incubate at room temperature for 5 min.
4. Put on magnetic rack, keep for 3 min. Remove supernatant.
5. Wash twice with 200 μl Ethanol 80% for 30 sec.
6. Carefully remove supernatant, leave tubes open for 10 min.
7. Resuspend beads in 42 µl of H2O, put on magnetic rack for 5 min. and collect supernatant. Keep 1 µl for Fragment Analyzer. Quantify the library with a Qubit.
Sequencing library preparation (using the SMRTbell™ Template Prep Kit)
1. Prepare the following mix, keeping the reagents on ice.
ds DNA: up to 37 µl
DNA Damage Repair Buffer: 5µl
NAD+: 0.5 µl
ATP high: 0.5 µl
dNTP: 0,5 µl
DNA Damage Repair Mix: 2 µl
water: __ µl to adjust
Tot. vol.: 50µl
2. Incubate at 37°C for 60 min.
3. Add 2.5 µl End Repair Mix, incubate at 25°C for 5 min.
4. Add 0.6X AMPure beads, incubate at room temperature for 10 min in a vortex mixer at 2000 rpm.
5. Spin down (1 sec.), put on magnetic rack to collect the beads and pipette off cleared supernatant.
6. Wash twice with 500 μL Ethanol 80% for 30 sec.
7. Spin down (1 sec.), carefully remove supernatant, leave tubes open for 10 sec in order to dry.
8. Resuspend beads in 30 μl EB, incubate at room temperature for 5 min. in a vortex mixer at 2000 rpm.
9. Spin down (1 sec.), put on magnetic rack for 1 min. and collect cleared supernatant (optional: keep 1 µl for Bioanalyzer).
10. For adapter ligation, prepare the following mix, keeping the reagents on ice.
DNA (End Repaired): 29 µl
Annealed Blunt Adapter: 2 µl
→ mix before processing
Template Prep Buffer: 4 µl
ATP low: 2 µl
→ mix before processing
Ligase: 1 µl
Water: 2 µl
Tot. vol.: 40 µl
11. Incubate at 25°C for 15 min., 65°C for 10 min.
12. For exonuclease treatment, add to 40 µl of ligated DNA:
Exo III: 0.5 µl
Exo VII: 0.5 µl
13. Incubate at 37°C for 60 min.
14. Add 0.6X AMPure beads, incubate at room temperature for 10 min in a vortex mixer at 2000 rpm.
15. Spin down (1 sec), put on magnetic rack to collect the beads and pipette off cleared supernatant.
16. Wash twice with 500 μl Ethanol 80% for 30 sec.
17. Spin down (1 sec), carefully remove supernatant, leave tubes open for 10 sec in order to dry.
18. Resuspend beads in 30 μl EB, incubate at room temperature for 5 min in a vortex mixer at 2000 rpm.
19. Spin down (1 sec), put on magnetic rack for 1 min. and collect cleared supernatant.
20. Repeat steps 14.-17.
21. Resuspend beads in 11 μl EB, incubate at room temperature for 5 min. in a vortex mixer at 2000 rpm.
22. Spin down (1 sec.), put on magnetic rack for 1 min. and collect cleared supernatant.
23. Check library quality on Bioanalyzer and Qubit.
Sample setup and sequencing
1. Use the Sample Setup module in SMRT Link to generate a customized pipetting protocol for primer conditioning, primer annealing and polymerase binding.
2. prepare the following mix:
Sequencing Primer v3: 1 µl
1x Elution buffer: 29.1 µl
3. Incubate at 80°C for 2 min., hold at 4°C.
Annealing Primer:
4. Mix (according to the customized pipetting protocol) the following:
sample
10x Primer buffer v2
conditioned Sequencing primer v3
water
5. Incubate at 20°C for 60 min, put on ice for immediate use or store at 20°C for long-term use.
6. Mix (according to the customized pipetting protocol) the following:
sample
Sequel dNTP
DTT
Sequel binding buffer
diluted Sequel Polymerase 3.0
water
7. Incubate at 30°C for 60 min., use bound complex right away or store at 4°C for up to 7 days.
8. Dilute the complex with Magbead binding buffer v2 (according to the customized pipetting protocol) and add AMPure beads; incubate at room temperature for 5 min.
9. Spin down (1 sec), put on magnetic rack to collect the beads and pipette off cleared supernatant. Important: do not wash with ethanol.
10. Resuspend beads in Magbead binding buffer v2, incubate at room temperature for 15 min.
11. Spin down (1 sec), put on magnetic rack for 1 min. and collect cleared supernatant.
12. Measure recovered DNA with Qubit and enter concentration and volume into the Sample Setup Module.
13. Mix the following (85 µl total volume per SMRT cell)
prepared sample
DTT
Diluted internal control
Sequel Additive
Sequel Complex Dilution buffer
14. Load 85 µl and store up to 24 h at 4°C.
15. Use the Run Design module in SMRTLink to create a run.
16. Refer to the table for loading and pre-extension recommendations for your insert size: https://www.pacb.com/wp-content/uploads/Quick-Reference-Card-Loading-and-Pre-Extension-Recommendations-for-the-Sequel-System.pdf
17. Set movie recording time to 360 - 600 min.; loading method: diffusion mode.
Data collection and analysis
Use the SMRT Analysis module in SMRTLink to create CCS fastq files (Minimum Number of Passes = 3, default parameters).
Once the raw data have been processed, the final CCS fastq files can be used directly as input for the FLAM-seq analysis pipeline, available at:
https://github.com/rajewsky-lab/FLAMAnalysis.