Extract and purify RNA from Brain tissue homogenate using .

Prepare the master mix for cDNA Prep as follows in a PCR plate/ tubes for 10μL reaction.

A	B
Component	Volume
Lunascrpit RT Supermix (5X)	2µl
Template	8µl
Total	10µl

Note

Prepare all reagents in a biosafety cabinet designated for clean work.

Incubate the reaction mix following the conditions mentioned below in a thermo cycler:

25°C for 00:02:00

55°C for 00:10:00

95°C for 00:01:00

Hold at 4°C

Primers are designed using Primal Scheme. Resuspend the lyophilized oligos fully in Nuclease free water / 1X TE to a concentration of 100µM, vortex thoroughly,Nuclease-free, and spin down.

RABV_Arctic_primers.csv

Separate odd and even primer sets. Add 5µl of each odd primer to a 1.5mL LoBind tube labelled Pool 1. Repeat the same for all even primers for Pool 2. These are the 100µM stocks of each primer pool.

Dilute 100µM stock pools (1:10) in Nuclease free water, to generate 10µM primer stock. A final concentration of 15 nM per primer is used. Here we use a total of 39 primers in Pool 1 and 39 primers in Pool 2. Here 0.75µl of per primer pool (10µM) is required for a 25µl reaction.

Set up two PCR reactions per sample (for Pool 1 and 2) as follows in tubes or plates. Mix by gentle pipetting and pulse spin to collect liquid at the bottom.

A	B	C
Component	Reaction Mix 1	Reaction Mix 2
5X Q5 Reaction Buffer	5µL	5µL
10 mM dNTPs	0.5µL	0.5µL
Q5 Hot Start DNA Polymerase	0.25µL	0.25µL
Pool 1 (10µM)	0.75µL	0µL
Pool 2 (10µM)	0µL	0.75µL
Nuclease-free water	18.5µL	18.5µL

Add 2.5µl cDNA to each of the PCR reactions, mix by gentle pipetting and spin the tube to collect all liquid at the bottom of the tube.

Set up the following PCR conditions in the thermal cycler.

Stage °C Cycles

Heat Activation 98 °C 00:00:30 1

Denaturation 98 °C 00:00:15 25-35

Annealing 65 °C 00:05:00 25-35

Hold 4 °C Indefinite 1

Label the tubes/plates and pool the PCR reactions for each samples into a single LoBind tube .

A	B
PCR pool 1	25µL
PCR pool 2	25µL
Nuclease Free water	0µL
Total	50µL

Clean-up the amplicons using .

Thaw the AMPure beads to room temperature and vortex thoroughly to ensure that the beads are well suspended/ until the solution is uniformly brown in colour.

Add 0.4x volume (0.4:1) of AMPure beads to the sample tube and mix gently by either flicking or pipetting. For example add 20,µl AMPure beads to a 50 µl reaction.

Quantify 1µl product using the Qubit Fluorometer using the .

Spin the tubes to collect entire liquid at the bottom and incubate the tubes at room temperature for 00.05.00.

Place the tubes on magnetic stand and incubate for 00:02:00 or until the beads have settled and the supernatant is completely colourless.

Carefully remove and discard the supernatant, without disturbing the bead pellet.

Add 200µl of 70% ethanol or enough to cover the pellet and wait for 30s.Carefully remove and discard ethanol delicately without disrupting the bead pellet.

and repeat Spin to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible.

Incubate the tube with open lid for 00:00:30 or until the pellet is dried (if the pellet dries completely it will crack and become difficult to resuspend)

Resuspend pellet in 30µl Elution Buffer (EB), mix gently by either flicking or pipetting and incubate for 00:02:00.Make sure that the pellet is completely resuspended.

Place on magnetic stand and transfer sample to a clean 1.5mL LoBind tube ensuring no beads are transferred into this tube.

In a new PCR plate/ tubes set up the following reaction for each sample. Prepare Master mix and aliquote it into tubes . If the Qubit reading is > 15ng/ul ,

A	B
Component	Volume
PCR dilution from previous step	3.3µL
Ultra II End Prep Reaction Buffer	1.2µL
Ultra II End Prep Enzyme Mix	0.5µL
Nuclease-free water	5µL
Total	10µL

If the Qubit reading is 5ng/ul -15ng/ul

A	B
Component	Volume
PCR dilution from previous step	5µL
Ultra II End Prep Reaction Buffer	1.2µL
Ultra II End Prep Enzyme Mix	0.5µL
Nuclease-free water	3.3µL
Total	10µL

If the Qubit reading is <5ng/ul,

A	B
Component	Volume
PCR dilution from previous step	8.3µL
Ultra II End Prep Reaction Buffer	1.2µL
Ultra II End Prep Enzyme Mix	0.5µL
Nuclease-free water	0µL
Total	10µL

Reaction conditions for End Prep:

Incubate

25°C for 00:15:00

65°C for 00:15:00

On ice for 00:01:00

In a new labelled tube/plate prepare the following components.

A	B
Component	Volume
End-preparation reaction mixture	0.75µL
NBXX barcode	1.25µL
Blunt/TA Ligase Master Mix	5µL
Nuclease-free water	3µL
Total	10µL

Incubate at

25°C for 00:20:00

65°C for 00:10:00

On ice for 00:01:00

Resuspend pellet in 30µl Elution Buffer(EB), mix gently by either flicking or pipetting and incubate for 00:02:00.

Place on magnet stand and transfer sample to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.

Quantify 1µl of the barcoded amplicons using the Qubit Flurometer.

Equipment

Value	Label
Qubit	NAME
Flurometer	TYPE
Invitrogen	BRAND
Q33228	SKU

In a new 1.5mL LoBind tube pool all barcoded reactions together.

Add 0.4x volume of AMPure beads to the sample tube and mix by either flicking or gentle pipetting. For example add 96µl AMPure beads to 240µl pooled barcoding reactions.

Briefly vortex and mix the reactions and incubate at room temperature for 00.05.00.

Place on magnetic stand and incubate for 00:02:00 or until the beads have pelleted and the supernatant is completely clear. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.

Remove the tube from the magnetic stand and add 200µl SFB and resuspend beads completely using pipette. Spin down to collect liquid at the bottom of the tube and place on the magnetic stand. Remove supernatant and discard.

Add 200µl of 70% ethanol to wash the pellet. Carefully remove and discard ethanol, being careful not to touch the bead pellet.

Spin to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible.

With the tube lid open incubate for 00:00:30 or until the pellet is dried (if the pellet dries completely it will crack and become difficult to resuspend).

Prepare the following reaction for AMII Adapter ligation in a PCR tube.

A	B
Component	Volume
Barcoded amplicon pool	30µL
NEBNext Quick Ligation Reaction Buffer (2X)	40µL
Adapter Mix (AMII)	5µL
Quick T4 DNA Ligase	5µL
Total	80µL

Incubate at 25°C for 00:20:00, possibly in a thermo cycler.

Add 80µl (1:1) of AMPure beads to the sample tube and mix gently by either flicking or pipetting. Spin to collect all liquid at the bottom of the tube.

Incubate for 00:05:00 at room temperature.

Place on magnetic rack and incubate for 00:02:00 or until the beads have pelleted and the supernatant is completely clear. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.

Add 200µl SFB and resuspend beads completely by pipette mixing. Brief spin to collect all liquid at the bottom of the tube. Remove supernatant and discard.

Repeat step 14.3 to perform a second SFB wash.

Brief spin and remove any residual SFB. Add 15µl EB (ONT) and resuspend beads by gentle flicking or pipette mixing.

Incubate at room temperature for 00:02:00 and place on magnetic stand until clear. Transfer final library to a new 1.5mL LoBind tube

Quantify 1µl of the barcoded amplicons using the Qubit Fluorometer.

Equipment

Value	Label
Qubit	NAME
Flurometer	TYPE
Invitrogen	BRAND
Q33228	SKU

Thaw the following reagents.

Sequencing buffer (SQB)

Loading beads (LB)

Flush buffer (FB)

Flush tether (FLT)

Add 30µl FLT to the FB tube and mix well by vortex and spin down.

Prepare the following library dilution for sequencing in a LoBind tube:

A	B
SQB	37.5µL
LB	25.5µL
Library	12-14µL
Total	75-80µL

Flowcell Priming, loading of the library and sequencing are performed based on the following protocol:

dx.doi.org/10.17504/protocols.io.bbmuik6w