Dec 12, 2022
Synthesis of double-strand cDNA (ds-cDNA) from viral dsRNA by using Random primers
- 1Département de Biologie, Centre SÈVE, Université de Sherbrooke, Sherbrooke, QC J1K 2R1, Canada
Protocol Citation: Vahid Jalali Javaran 2022. Synthesis of double-strand cDNA (ds-cDNA) from viral dsRNA by using Random primers. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbyrxnvpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 12, 2022
Last Modified: December 12, 2022
Protocol Integer ID: 73881
Abstract
Double-stranded cDNA synthesis from viral dsRNAs:
For dsRNA sequencing by nanopore sequencing, this protocol was used. Before treating samples with RNase T1, you should measure the total concentration of RNAs in the samples by using a nanodrop or Qubit device, as RNase T1 has the ability to partially digest double-stranded RNAs in the absence of single-stranded RNA.
Materials
RNase T1 and DNase I digestion
- DNase I (RNase-free)
- DNase I Reaction Buffer with MgCl2 (10X)
- RNase T1
Synthesize the first strand of cDNA (Reverse Transcription)
- Random primers (60 µM)
- dNTP (10 mM)
- H2O
- First Strand cDNA synthesis Buffer
- RNase out or RNasin® Ribonuclease Inhibitor (40 u/µl)
- Superscript III or Maxima H minus
Removal of the residual RNA by RNase H
- RNase H
Synthesize the second strand of cDNA
- Klenow 10X buffer+NAD,
- dNTP (10 mM),
- Klenow DNA Polymerase I,
- E. coli DNA Ligase I
cDNA purification by AMPure XP beads
- Agencourt AMPure XP
- Magnet plate
- Ethanol 80%
- Nuclease free water
- 1.5 ml eppendorf tube
RNase T1 and DNase I digestion
RNase T1 and DNase I digestion
1h 20m
1h 20m
- Add 10X DNase Buffer with MgCl2 (final concentration should be 1X).
- Add 50 units RNase T1 per 1µg of total RNA and 1 unit DNase I per 2ug of total RNA.
- Incubate at 37 degrees C for 20 min.
20m
Stop reaction
Do a phenol-chloroform extraction and Ethanol precipitation of dsRNA.
1h
Synthesis of the first strand of cDNA
Synthesis of the first strand of cDNA
2h
2h
Mix well below components by pipetting and centrifuge or spin briefly.
| A | B | |
| Treated dsRNA | 5 µl | |
| Random primers (60 µM) | 2µl | |
| dNTP (10 mM) | 1 µl | |
| H2O | 6 µl | |
| Total | 14 µl |
- Incubate at 99C for 5 mins and put tubes immediately on water ice (it is better to do this step with a thermocycler).
5m
Add below components and mix well by pipetting and centrifuge or spin briefly.
| A | B | |
| First Strand Buffer | 4 µl | |
| RNase out or RNasin® Ribonuclease Inhibitor (40 u/µl) | 1 µl | |
| Maxima H minus (Point: just use 200 unit) | 1 µl | |
| Total | 20 µl |
Incubation step.
| A | B | |
| 25 C | 20 mins | |
| 55 C | 90 mins | |
| 85 C (Inactivation step) | 5 mins |
1h 55m
Removal of residual RNA by RNase H
Removal of residual RNA by RNase H
20m
20m
Mix well below components by pipetting and centrifuge or spin briefly.
| A | B | |
| cDNA | 20 µl | |
| RNase H | 1 µl | |
| Total | 21 µl |
Incubate at 37 C for 20 mins
20m
Synthesis of the second strand of cDNA
Synthesis of the second strand of cDNA
2h 40m
2h 40m
Mix well below components by pipetting and centrifuge or spin briefly.
| A | B | |
| cDNA | 21 µl | |
| Klenow 10X buffer | 2.7 µl | |
| dNTP (10 mM) | 1 µl | |
| Klenow DNA Polymerase I | 0.7 µl | |
| E. coli DNA Ligase I | 1 µl | |
| H2O | 0.6 µl | |
| Total | 27 µl |
Incubation step.
| A | B | |
| 16 C | 150 mins | |
| 75 C; (Inactivation step) Point: check this part based on enzyme brand. | 10 mins |
2h 40m
cDNA purification by AMPure XP
cDNA purification by AMPure XP
17m
17m
Mix well below components by pipetting and incubate at room temperature for 5 mins.
Point: Put bead stock in room temperature for 20 min (before using). Vortex the bead stock very well (before using)
| A | B | |
| double stranded-cDNA | 27 μL | |
| AMPure XP | 49 μL |
Place the reaction tube onto a magnet plate for 2-5 mins
IMPORTANT: Wait for the solution to clear before proceeding to the next step.
7m
This step must be performed while the reaction tube is situated on the magnet plate:
- Aspirate the cleared solution from the reaction tube and discard. Leave 5 μL of supernatant behind, otherwise, beads are drawn out with the supernatant.
IMPORTANT: Do not disturb the ring of separated magnetic beads.
This step must be performed while the reaction tube is situated on the magnet plate:
Dispense 200 μL of 80% ethanol to the reaction tube and incubate for 30 seconds at room temperature. Aspirate out the ethanol and discard.
Repeat previous step.
1m
After removing and discarding all supernatant, A dry time is optional (2 to 5 mins) to ensure all traces of Ethanol are removed.
NOTE: For fragments 10 kb and larger, do not over-dry the bead ring (bead ring appears cracked if over dried) as this will significantly decrease elution efficiency.
2m
Remove the reaction tube from the magnet plate, and then add 40 μL of nuclease-free water to the reaction tube and pipette mix 10 times. Incubate for 2 minutes.
NOTE The liquid level will be high enough to contact the magnetic beads at a 40 μL nuclease-free water. A greater volume of nuclease-free water can be used, but using less than 40 μL will require extra mixing (to ensure the liquid comes into contact with the beads), and may not be sufficient to elute all cDNAs.
2m
Place the reaction tube onto the magnet plate for 5 minute to separate beads from the solution.
IMPORTANT Wait for the solution to clear before proceeding to the next step.
Transfer the eluate to a new plate.
Use 1 μL of eluted ds-cDNA for concentration measurement by Qubit.
5m