Dec 12, 2022

Public workspaceSynthesis of double-strand cDNA (ds-cDNA) from viral dsRNA by using Random primers V.1

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l69nddlqe/v1
Synthesis of double-strand cDNA (ds-cDNA) from viral dsRNA by using Random primers
  • Vahid Jalali Javaran1
  • 1Département de Biologie, Centre SÈVE, Université de Sherbrooke, Sherbrooke, QC J1K 2R1, Canada
  • Nanovirseq
Vahid Jalali Javaran
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l69nddlqe/v1
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.bp2l69nddlqe/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: September 24, 2022
Last Modified: December 12, 2022
Protocol Integer ID: 70472
Keywords: cdna synthesis from viral dsrna, samples with rnase t1, rnase t1, viral dsrna, stranded cdna synthesis, cdna synthesis, stranded rna, rna, total concentration of rna, strand cdna, rnas in the absence, nanopore, d
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
Troubleshooting
RNase T1 and DNase I digestion
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
2h
Mix well below components by pipetting and centrifuge or spin briefly.
AB
Treated dsRNA5 µl
Random primers (60 µM)2µl
dNTP (10 mM)1 µl
H2O 6 µl
Total14 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.
AB
First Strand Buffer4 µl
RNase out or RNasin® Ribonuclease Inhibitor (40 u/µl)1 µl
Maxima H minus (Point: just use 200 unit)1 µl
Total20 µl

Incubation step.
AB
25 C20 mins
55 C90 mins
85 C (Inactivation step)5 mins
1h 55m
Removal of residual RNA by RNase H
20m
Mix well below components by pipetting and centrifuge or spin briefly.
AB
cDNA20 µl
RNase H1 µl
Total21 µl
Incubate at 37 C for 20 mins
20m
Synthesis of the second strand of cDNA
2h 40m
Mix well below components by pipetting and centrifuge or spin briefly.
AB
cDNA21 µl
Klenow 10X buffer2.7 µl
dNTP (10 mM)1 µl
Klenow DNA Polymerase I0.7 µl
E. coli DNA Ligase I1 µl
H2O0.6 µl
Total27 µl
Incubation step.
AB
16 C150 mins
75 C; (Inactivation step) Point: check this part based on enzyme brand.10 mins
2h 40m
cDNA purification by AMPure XP
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)
AB
double stranded-cDNA27 μL
AMPure XP49 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