TITLE: Synthesis of double-strand cDNA (ds-cDNA) from viral dsRNA by using Random primers
AUTHORS: Vahid Jalali Javaran

[DESCRIPTION]
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.


[STEPS]
SECTION: Synthesis of the first strand of cDNA
17. Mix well below components by pipetting and centrifuge or spin briefly. 
Treated dsRNA5 µlRandom primers (60 µM)2µldNTP (10 mM)1 µlH2O 6 µlTotal14 µl

SECTION: RNase T1 and DNase I digestion
1. 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.
SECTION: Synthesis of the first strand of cDNA
2. Mix well below components by pipetting and centrifuge or spin briefly.
 
 
        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). 
SECTION: RNase T1 and DNase I digestion
1.1. Stop reaction
Do a phenol-chloroform extraction and Ethanol precipitation of dsRNA.
SECTION: Synthesis of the first strand of cDNA
2.1. Add below components and mix well by pipetting and centrifuge or spin briefly.
 
      First Strand Buffer  4 µl    RNase out or RNasin® Ribonuclease   Inhibitor (40 u/µl)  1 µl    Maxima H minus  1 µl    Total  20 µl  

SECTION: Synthesis of the first strand of cDNA
2.2. Incubation step.
 
      25 C  20 mins    55 C  90 mins    85 C (Inactivation step)  5 mins  
 
SECTION: Removal of residual RNA by RNase H
3. Mix well below components by pipetting and centrifuge or spin briefly.
 
      cDNA  20 µl    RNase H  1 µl    Total  21 µl  
 Incubate at 37 C for 20 mins
SECTION: Synthesis of the second strand of cDNA
4. Mix well below components by pipetting and centrifuge or spin briefly.
 
      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    Water  0.6 µl    Total  27 µl  
 
SECTION: Synthesis of the second strand of cDNA
4.1. Incubation step.
 
      16 C  150 mins    75C    (Inactivation step) Point: check this part based on enzyme brand)  10 mins  
 
SECTION: cDNA purification by AMPure XP
5. 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)
 
      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.

SECTION: cDNA purification by AMPure XP
5.1. 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.

SECTION: cDNA purification by AMPure XP
5.2. 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.

SECTION: cDNA purification by AMPure XP
5.3. 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.

SECTION: cDNA purification by AMPure XP
5.4. 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.

SECTION: cDNA purification by AMPure XP
5.5. 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.