TITLE: Optimized conditions for whole genome sequencing of avian reoviruses.
AUTHORS: Sonsiray Alvarez-Narvaez, telvin_harrell, s.conrad steven_j_conrad

[DESCRIPTION]
Whole genome sequencing (WGS) is becoming an essential tool to characterize the genomes of avian reovirus (ARV), an emerging pathogen and growing threat to poultry producers and integrators. This protocol details a workflow for ARV  genome purification and amplification to increase the yield of ARV reads after next generation sequencing.


[GUIDELINES]
This protocol is per 6 x 106cells (2xT75 flasks at 95 % confluency) infected with approximately 20 μL of ARV supernatant and incubated (38°C, humidified, with 5% CO2) for 5 days, but it is scalable to larger or smaller cell lysates. Adjust volumes according to relative number of cells

[STEPS]
SECTION: RNA extraction
7. We are going to use MagMAX™ Viral RNA Isolation Kit (Applied Biosystems, catalog number AM1939) to extract the RNA from the purified ARV virions.

        NOTE: Spray work surfaces and pipettes with an RNase decontamination solution and wipe         clean.
SECTION: Host rRNA depletion
13. Preparing custom probes

This part of the protocol was adapted from the original protocol detailed in Parris, D.J., H. Kariithi, and D.L. Suarez, Non-target RNA depletion strategy to improve sensitivity of next-generation sequencing for the detection of RNA viruses in poultry. J Vet Diagn Invest, 2022. 34(4):p. 638-645.
The sequence of the probes can be found in the manuscript.

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13.1. Dilute the probes to 100 µM stocks.
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19. Reverse Transcription
We are going to use SuperScript IV Reverse Transcriptase (ThermoFisher Scientific, catalog number 18090010) to perform the reverse transcription step.
SECTION: ARV virion purification
5. Preparing 50% Capto700 Slurry
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5.1. Spin CaptoCore700 resin at 800 x g for 5 minutes.
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5.2. Remove as much supernatant as possible without disturbing the resin.
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5.3. Record the volume of resin left in the tube.
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5.4. Add equal-to-resin volume of VDB and mix by vortexing. 
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5.5. Repeat steps 5.1 to 5.4.
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5.6. Store as "50% slurry in VDB" at room temperature.
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6. Virion purification
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6.1. Centrifuge the infected cell lysates at 800 x g for 10 minutes to remove nuclei and debris.
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6.2. Collect the supernatant in a fresh tube containing 100 µl of 50% CaptoCore 700 Slurry.
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6.3. Mix sample end-over-end for 45 minutes at room temperature.
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6.4. Subject sample to centrifugation at 800 x g for 10 minutes.
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6.5. Transfer the top phase to a new tube containing 100 µl of 50% CaptoCore 700 Slurry.
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6.6. Repeat steps 6.2 to 6.5 for increasing purity.
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6.7. To clear all CaptoCore 700 resin, pass the supernatant of the last cleaning cycle through an Illustra MicroSpin column at 800 x g for 5 minutes.
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6.8. Store virus at 4ºC, or aliquot and store at -20ºC for long-term usage. Avoid freeze-thaw cycles.
SECTION: Introduction
1. NOTES:
This protocol is per 6 x 106cells (2xT75 flasks at 95 % confluency) infected with approximately 20 μL of ARV supernatant and incubated (38°C, humidified, with 5% CO2) for 5 days, but it is scalable to larger or smaller cell lysates. Adjust volumes according to relative number of cells

SECTION: ARV collection
2.1. List of reagents:
 

9. The sequence of the probes can be found in Parris, D.J., H. Kariithi, and D.L. Suarez, Non-target RNA depletion strategy to improve sensitivity of next-generation sequencing for the detection of RNA viruses in poultry. J Vet Diagn Invest, 2022. 34(4):p. 638-645.

Dilute the probes to 100uM stocks. 
For working master mix for the depletion
protocol, pool equal volumes (1 µL) of each probe.
SECTION: cDNA production and single primer amplification
20. cDNA second strand synthesis
We are going to use Klenow polymerase (NEB,product number M0210S) to generate double-stranded (ds) cDNA.

SECTION: cDNA production and single primer amplification
19.1. Spray work surfaces with Eliminase (or similar cleaner) and wipe clean.
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19.2. Combine the following on a reaction tube:
 

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19.3. Add 10 μl of the extracted RNA to the reaction tube for a final volume of 13 μl.
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19.4. Mix briefly by carefully pipetting up and down 10 times
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19.5. Heat up the RNA-primer mix at 95ºC for 4 mins and incubate one ice for 1 min
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19.6. Prepare the SuperScript IV reaction (7 μl vol.):
 

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19.7. Mix briefly by carefully pipetting up and down 10 times
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19.8. Add the SuperScript IV reaction (7 μl) to the RNA-primer mix (13 μl) and mix well by pipetting up and down 20 times.
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19.9. Incubate the 20 μl reaction in the thermocycler under the following conditions:
 
NOTE: Use your first-strand cDNA immediately  to generate double-stranded cDNA or store at -20 ºC.

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20.1. Make a stock of primer R8N to 10 μM



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20.2. Add the following to the 20μl reverse transcriptase reaction:

 

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20.3. Mix well by carefully pipetting up and down 20 times
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20.4. Heat up the mix at 94ºC for 3 mins and cool down on at 4 ºC in the thermocycler
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20.5. Add 1 ul of Klenow fragment and mix well by carefully pipetting up and down 20 times
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20.6. Incubate the mix at 37 °C for 60 min (final volume, 25 μl)
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21. Double-stranded cDNA purification
We are going to use Agencourt AMPure XP beads (Beckman Coulter, product number A63880) to purify ds-cDNA
    
            Before you start: Bring AMPure XP beads to room temperature (RT) at least 30 mins before starting purification. Vortex RNA sample purification beads to resuspend. Prepare 70% ethanol (nuclease free).  Mix 7 parts 100% ethanol with 3 parts nuclease-free H2O. You will need 600ul 70% ethanol per sample.
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21.1. Vortex the Agencourt AMPure XP bottle to resuspend any magnetic particles that may have settled.
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21.2. Add 45 μl AMPure XP beads (X 1.8) to the 25 μl ds-cDNA reaction
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21.3. Mix well by carefully pipetting up and down 20 times. The color of the mixture should appear homogenous after mixing.
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21.4. Let the mixed samples incubate for 5 minutes at RT.
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21.5. Place the reaction plate onto the magnet for 2 minutes to separate beads from the solution. Wait for the solution to clear before proceeding to the next step.
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21.6. Carefully remove and discard the supernatant (cleared solution).
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21.7. With your tubes still on the magnet stand, add 200 μl of 70% ethanol to each well and incubate for 30 seconds at RT. Then, carefully remove and discard the ethanol.
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21.8. Repeat step 21.7 for a total of two washes.
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21.9. Remove traces of ethanol with a 20 μl pipette and air dry the beads for up to 5 mins at RT.
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21.10. Remove the reaction plate from the magnet plate, and then add 20 μL of nuclease-free H2O to each well of the reaction plate and pipette mix 10 times. Incubate for 2 minutes at RT.
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21.11. Place the reaction plate back onto the magnet and wait for 1 minute (or until the solution is clear) to separate beads from the solution.
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21.12. Transfer 15 μL the supernatant containing your clean ds-cDNA (clear solution) into a new tube.
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22. ds-cDNA amplification by PCR
We are going to use a HiFi PCR Phusion kit (NEB, catalog number M0530S) for ds-cDNA PCR amplification.



SECTION: cDNA production and single primer amplification
22.1. Prepare the PCR reaction as follows (final vol. 45 μl):
 

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22.2. Mix well by carefully pipetting up and down 10 times
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22.3. Add 5 μl of clean ds-cDNA to each PCR reaction mix to have a final volume of 50 μl.
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22.4. Place the 50 μl reaction in a thermocycler and run the PCR as follows:

 

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21.13. NOTE: Use your ds-cDNA immediately  to generate double-stranded cDNA or store at -20 ºC.
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23. PCR product purification
We are going to use Agencourt AMPure XP beads (Beckman Coulter, product number A63880) to purify ds-cDNA
    

            Before you start: Bring AMPure XP beads to RT at least 30 mins before starting purification. Prepare 70% ethanol (nuclease free).  Mix 7 parts 100% ethanol with 3 parts nuclease-free H2O. You will need 600ul 70% ethanol per sample.

SECTION: cDNA production and single primer amplification
23.1. Vortex the Agencourt AMPure XP bottle to resuspend any magnetic particles that may have settled.
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23.2. Add 90 μl AMPure XP beads (X 1.8) to the 50 μl PCR product
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23.3. Mix well by carefully pipetting up and down 20 times. The color of the mixture should appear homogenous after mixing.
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23.4. Let the mixed samples incubate for 5 minutes at RT.
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23.5. Place the reaction plate onto the magnet for 2 minutes to separate beads from the solution. Wait for the solution to clear before proceeding to the next step.
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23.6. Carefully remove and discard the supernatant (cleared solution).
SECTION: cDNA production and single primer amplification
23.7. With your tubes still on the magnet stand, add 200 μl of 70% ethanol to each well and incubate for 30 seconds at RT. Then, carefully remove and discard the ethanol.
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23.8. Repeat step 23.7 for a total of two washes.
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23.9. Remove traces of ethanol with a 20 μl pipette and air dry the beads for up to 5 mins at RT.
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23.10. Remove the reaction plate from the magnet plate, and then add 35 μL of nuclease-free H2O to each well of the reaction plate and pipette mix 10 times. Incubate for 2 minutes at RT.
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23.11. Place the reaction plate back onto the magnet and wait for 1 minute (or until the solution is clear) to separate beads from the solution.
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23.12. Transfer 30 μL the supernatant containing your clean ds-cDNA (clear solution) into a new tube.
SECTION: ARV collection
3.1. Harvest infected LMH cells in their own supernatant with a cell scraper (Corning™ catalogue number 
3011) and centrifuged at 3000 x g for 10 mins at room temperature (RT).

SECTION: ARV collection
3.3. Sonicate the infected cells on ice: 3 pulses at 30% amplitude, 10 sec. on, 30 sec. off, we used a Branson Digital Sonifier 450 (Branson Ultrasonics Corporation).
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3.2.   Resuspended the pellet in 350 µL of VDB.
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3.4. Store the infected cell lysates for not more than a week at 4ºC.
SECTION: RNA extraction
8. Prepare Lysis/Binding Solution

 

8. 
SECTION: Host rRNA depletion
13.2. Pool equal volumes (1 µl) of each probe to make the working master mix for the depletion
protocol.
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13.3. Aliquot the master mix depending on the  number of samples that are going to be frequently sequenced

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14.1. Prepare RNA-Probe master mix per sample
 

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14.2. Add 3 µl of the above mix to 12 µl of extracted RNA (final vol. 15 µl)
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14.3. Mix by pipetting up and down  10 times
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14.4. Spin down briefly in a tabletop centrifuge to collect the volume to the bottom of the tube.
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14.5. Place samples in a thermocycler with a heated lid set to 105°C, and run the following program:

 

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14. ssDNA probes hybridization
We are going to hybridize a set of custom probes to the chicken rRNA and mitochondrial RNA.

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14.6. Place samples on ice and proceed immediately to the next step.



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15. RNase H digestion
We are going to use RNase H enzyme (New England Biolabs, catalogue number M0297S) to degrade chicken and bacteria rRNA that hybridized with the ssDNA probes.
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15.1. On ice, prepare the following master mix and use immediately
 

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15.2. Add 5 µl of the master mix to the RNA sample (15 µl) from step 14.6 (final vol. 20 µl).
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15.3. Mix by pipetting up and down 10 times.
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15.4. Spin down briefly in a tabletop centrifuge to collect the volume to the bottom of the tube.
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15.5. Place samples in a thermocycler with heated lid off) and incubate at 37°C for 30 min.
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15.6. Place samples on ice and proceed immediately to the next step.
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16. DNase I Digestion
We are going to use DNase I enzyme (New England Biolabs, catalog number M0303S) to degrade the custom ssDNA probes from the samples.
SECTION: Host rRNA depletion
16.1. On ice, prepare a DNase I Digestion Master Mix 

 

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16.4. Mix by pipetting up and down 10 times.
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16.5. Spin down briefly in a tabletop centrifuge to collect the volume to the bottom of the tube.
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16.6. Place samples in a thermocycler with heated lid off) and incubate at 37°C for 30 min.
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16.7. Place samples on ice and proceed immediately to the next step.
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16.3. Add 30 µl of the above mix to the RNA sample from Step 15.6 (final vol. 50 µl).
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16.2. Mix by pipetting up and down 10 times.
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17. Bead-purification of RNAs
We are going to use RNAClean XP beads (Beckman Coulter, catalog number A63987) to clean the RNA before cDNA synthesis.


Before you start: Bring  RNAClean XP  beads to room temperature (RT) at least 30 mins before starting purification. Vortex RNA sample purification beads to resuspend. Prepare 80% ethanol (nuclease free).  Mix 7 parts 100% ethanol with 3 parts nuclease-free H2O. You will need 600ul 80% ethanol per sample.
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17.1. Add 110 μl (2.2X)  RNAClean XP beads to 50 µl to host depleted RNA.
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17.3. Incubate samples at RT for 15 min.
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17.4. Place the plate on magnetic stand to separate the beads from the supernatant.
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17.5. After 5 min (or when the solution is clear), carefully remove and discard the supernatant.
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17.6. Add 200 μl of 80% freshly prepared ethanol to the tube/ plate while in the magnetic stand. Incubate at RT for 30 seconds, and then carefully remove and discard the supernatant.
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17.7. Repeat Step 17.6. Place back on the magnet and remove traces of ethanol with a p10 pipette tip.
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17.8. Air dry the beads for up to 5 min while the plate is on the magnetic stand with the lid open. 
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17.9. Remove the plate from the magnetic stand and elute the RNA from the beads by adding 15 μl of nuclease-free H2O.
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17.10. Mix well by pipetting up and down 10 times. Incubate for 5 min. 
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17.12. Place the plate on the magnetic stand for 5 min.
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17.14. Place the tube on ice and proceed with cDNA production. Alternatively, the sample can be stored at –80°C.
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17.2. Mix by pipetting up and down 10 times.
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17.11. Fast-spin the sample to collect the liquid from the sides of the tube.
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17.13. Transfer 12 μl of the clean host-depleted RNA to a new PCR tube.
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23.13. Now your ARV cDNAs are ready for genomic library preparation. Alternatively, they can be stored at-20 ºC
SECTION: ARV collection
2. Prepare Virus Dilution Buffer (VDB)
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3. Preparing cell lysates
SECTION: ARV virion purification
4. This part of the protocol was adapted from the original protocol detailed in James, K.T., et al., Novel High-throughput Approach for Purification of Infectious Virions.Sci Rep, 2016. 6: p. 36826. 
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18. This part of the protocol was adapted from the original protocol detailed in Chrzastek, K., H. Sellers, and D. Kapczynski, A universal, single primer amplification protocol (R-SPA) to perform whole genome sequencing of segmented dsRNA reoviruses. 2021, bioRxiv. 
SECTION: RNA extraction
9. Prepare the Wash solution 1
Add 12 mL 100% isopropanol to the bottle labeled Wash Solution 1 Concentrate and mix well. Mark the label to indicate that the isopropanol was added. Store at room temperature.
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10. Prepare the Wash solution 2
Add 44 mL 100% ethanol to the bottle labeled as Wash Solution 2 Concentrate and
mix well. Mark the label to indicate that the ethanol was added. Store at room temperature.
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11. Prepare Bead Mix
Vortex the RNA Binding Beads at moderate speed to form a uniform suspension before pipetting. 

 

 Include ~10% overage to cover pipetting error when preparing the Bead Mix.
Mix thoroughly by gently vortexing.
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12. RNA extraction
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12.1. Add 400 µL sample to 802 µL Lysis/Binding Solution in a 2ml microcentrifuge tube. 
IMPORTANT: When adding sample, immerse pipette tips slightly in the Lysis/Binding Solution to prevent creating aerosols that can lead to cross-contamination.
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12.2. Mix by gently  flicking the tube several times.
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12.3. Centrifuge the mix briefly (~2 sec) to collect the contents at the bottom of the tube.
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12.4. Vortex the Bead Mix at moderate speed to create a uniform suspension before pipetting
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12.5. Add 20 µL Bead Mix to each sample
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12.6. Gently shake the sample for 4 min on a vortex mixer to fully lyse viruses and bind RNA to the RNA Binding Beads.
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12.7. Centrifuge the samples briefly (~2 sec) to collect tube contents.
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12.8. Move the samples to a magnetic stand to capture the RNA Binding Beads.
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12.9. Leave the tubes on the magnetic stand for 3 minutes or until a clear bead pellet is formed.
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12.11. Remove the Processing Tube from the magnetic stand.
12.12. Add 300 µL Wash Solution 1 to each sample and vortex at moderate speed for 30 sec.
12.13. Centrifuge briefly (~2 sec) to collect tube contents.
12.14. Leave the tubes on the magnetic stand for 5 minutes or until a clear bead pellet is formed.
12.15. Carefully aspirate and discard the supernatant without disturbing the beads.
12.16. Remove the Processing Tube from the magnetic stand.
12.17. Repeat steps 12.12 -12.15 to wash a second time with 300 µL Wash Solution 1.
12.18. Add 450 µL Wash Solution 2 to each sample and vortex at moderate speed for 30 sec.
12.19. Centrifuge briefly (~2 sec) to collect tube contents.
12.20. Leave the tubes on the magnetic stand for 5 minutes or until a clear bead pellet is formed.
12.21. Carefully aspirate and discard the supernatant without disturbing the beads
12.22. Remove the Processing Tube from the magnetic stand. 
12.23. Repeat steps 12.18 -12.21 to wash a second time with 450 µL Wash Solution 2.
12.24. Leave the tube open on the magnetic stand at room temp for 2 min to allow any remaining alcohol from the second wash with Wash Solution 2 to evaporate.
12.25. Inspect the tubes and if there is residual solution, remove as much as possible with a very fine-tipped pipettor.
12.26. Leave the tube open on the bench for another minute.
12.27. Add 30 µL Elution Buffer (room temp) to each sample, and shake or vortex vigorously for 4 min.
12.28. Leave the tubes on the magnetic stand for 5 minutes or until a clear bead pellet is formed.
12.29. Transfer 26uL of the supernatant, which contains the RNA, to a nuclease-free container appropriate for your application, and store the purified RNA at –20°C.
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12.10. Carefully aspirate and discard the supernatant without disturbing the beads.