TITLE: Sequencing the Virome of Wild Eastern Cottontail Rabbits using Oxford Nanopore Technologies (ONT)
AUTHORS: Paula Glover

[DESCRIPTION]
This protocol can be used to perform multiplex sequencing of a virome using Oxford Nanopore Technologies (ONT). 

Forty RNA samples, including blood (n = 10), fecal (n = 10), epithelial (n = 10) and mucosal (n = 10) collected using cotton swabs from 10 wild Eastern Cottontail rabbits and stored at -80 °C. The 40 samples were combined into four pools each composed of ten samples of the same type. Sequencing was performed using the cDNA-PCR kit from 

[STEPS]
SECTION: Pre-processing of Samples: Blood, Fecal, Epithelial, and Mucosal (Salival)
1.1. Place each swab in a 1.5-mL centrifuge tube with 500 µL of phosphate buffered saline (PBS). PBS is an isotonic solution that prevents the rupture of cells or shriveling of cells due to osmosis and is a non-toxic formulation for the cells.

SECTION: Pre-processing of Samples: Blood, Fecal, Epithelial, and Mucosal (Salival)
1.2. Vortex the samples for 5 min at room temperature, then centrifuge for 5 min at10000 rpm. Collect  400 µL  in a fresh 1.5 ml Eppendorf DNA LoBind tube. 

SECTION: Pre-processing of Samples: Blood, Fecal, Epithelial, and Mucosal (Salival)
2. Mucosal (Salival) Sample Pre-Processing (Dilution)
SECTION: Pre-processing of Samples: Blood, Fecal, Epithelial, and Mucosal (Salival)
2.1. Dilute 100 µL  of the sample in 300 µL of Saline Solution 0.8 Mass Percent in a fresh 1.5 ml Eppendorf DNA LoBind tube. Normal saline solution can serve as RNA preservative, eliminating the need for snap-freezing the sample.

SECTION: Pre-processing of Samples: Blood, Fecal, Epithelial, and Mucosal (Salival)
2.2. Vortex mix 10 s . 

SECTION: RNA Extraction Using the TRIzol™ Protocol by Invitrogen
3. Critical Step: Ensure you start with at least 3X more TRIzol‱ than the volume of your starting material. Extract 250 µLof each sample in a 1.5 ml Eppendorf DNA LoBind tube. 

SECTION: RNA Extraction Using the TRIzol™ Protocol by Invitrogen
4. Follow the standard TRIzol™ protocol, using a Phase Lock GelTM tube to trap the organic phase. Add 1 µg RNase-free glycogen to the first isopropanol precipitation. Then use standard 1.5 ml tubes for the pelleting steps.

SECTION: RNA Extraction Using the TRIzol™ Protocol by Invitrogen
4.1. Optional Step: GlycoBlue Coprecipitant can be used to make the pellet visible.
SECTION: RNA Extraction Using the TRIzol™ Protocol by Invitrogen
5. Elute the RNA in 100 µL nuclease-free water or TE buffer. (Yields 10-20 ng of RNA)

SECTION: Pre-processing of Samples: Blood, Fecal, Epithelial, and Mucosal (Salival)
1. Blood, Fecal, and Epithelial Sample Pre-processing
SECTION: Library Preparation with the Direct RNA Sequencing Kit (SQK-RNA004)
20. Use the Direct RNA Sequencing (SQK-RNA004)Protocol from Oxford Nanopore Technologies (ONT). The table below is an overview of the steps required in the library preparation, including timings and stopping points.

 

 

SECTION: Polyadenylating with E. coli poly(A) polymerase
7. In a 1.5 ml microcentrifuge tube, set up the 3’ polyadenylation reaction as follows:

 

SECTION: Polyadenylating with E. coli poly(A) polymerase
7.1. Incubate the reaction mixture at37 °C for 1 min .

SECTION: Polyadenylating with E. coli poly(A) polymerase
7.2. Stop the reaction by adding 5 µLof 50 mM EDTA (to a final concentration of 10 mM EDTA). The final volume will be 25 µL
SECTION: Polyadenylating with E. coli poly(A) polymerase
7.3. Note: An incubation time of 30–90 seconds to add a 3’ adenosine homopolymer (poly(A) tail) of approximately 50-100 nucleotides in length. An increase in incubation time will result in longer poly(A) tails. The maximum incubation time is 5 minutes and longer timings may result in a lower total sequencing yield. 
SECTION: Polyadenylating with E. coli poly(A) polymerase
7.4. Optional Step: It is recommended that EDTA be removed before starting any sequencing kit protocols.
SECTION: Polyadenylating with E. coli poly(A) polymerase
8. Add 45 µLof RNase-free SPRI beads to the reaction.

SECTION: Polyadenylating with E. coli poly(A) polymerase
9. Incubate on a Hula Mixer for 5 minat room temperature.

SECTION: Polyadenylating with E. coli poly(A) polymerase
10. Spin down the sample and pellet on a magnet.
SECTION: Polyadenylating with E. coli poly(A) polymerase
11. . Keep the tube on the magnet and pipette off and discard the supernatant.
SECTION: Polyadenylating with E. coli poly(A) polymerase
12. Keep the tube on the magnet and wash the beads with 200 µl of freshly-prepared 70% ethanol. Carefully turn the tube 180° twice in the rack to wash pelleted beads. Pippette off and discard the supernatant.
SECTION: Polyadenylating with E. coli poly(A) polymerase
13. Repeat the previous step for a total of two washes.
SECTION: Polyadenylating with E. coli poly(A) polymerase
14. Briefly spin down and place the tube back on the magnet. 
SECTION: Polyadenylating with E. coli poly(A) polymerase
14.1. Pipette off any residual ethanol. Allow to dry for 30 s, but do not dry the pellet to the point of cracking.
SECTION: Polyadenylating with E. coli poly(A) polymerase
15. Remove the tube from the magnetic rack and resuspend the pellet in 12 µL of nuclease-free water. Incubate on ice for 5 min.
SECTION: Polyadenylating with E. coli poly(A) polymerase
16. Pellet the beads on a magnet until the eluate is clear and colourless.
SECTION: Polyadenylating with E. coli poly(A) polymerase
17. Remove and retain 12 µL of eluate containing the 3’-polyadenylated RNA in a clean 1.5 ml microcentrifuge tube.
SECTION: Polyadenylating with E. coli poly(A) polymerase
18. Remove 1 µLof final eluate and quantify the final concentration using HS RNA Qubit kit.

SECTION: Polyadenylating with E. coli poly(A) polymerase
18.1. Optional step: Dilute the final eluate to 100 ng/µl and quantify 1 µL using Agilent 6000 Nano RNA kit, using total RNA or mRNA setting depending on the sample.

SECTION: Polyadenylating with E. coli poly(A) polymerase
19. Store the final product at -80 °C or proceed immediately with the library preparation, keeping your sample on ice.

SECTION: Assessing input RNA
6. Oxford Nanopore Technologies (ONT) protocols recommend an input quantity in mass. The Direct RNA Sequencing Kit (SQK-RNA004) from ONT is used to prepare RNA for nanopore sequencing from an input of as low as 300 ng poly(A)+ RNA or 1 µg of total RNA (additional optimization may be required for total RNA). Therefore, it is important to assess the input RNA to determine if it is of high enough quality and amount to be used in direct RNA sequencing. The following protocol is described by ONT for assessing input RNA:

SECTION: Assessing input RNA
6.1.