Mar 25, 2024
Nanopore SISPA
This protocol is a draft, published without a DOI.
- 1University of Hong Kong
Protocol Citation: Siu Fung Ho 2024. Nanopore SISPA . protocols.io https://protocols.io/view/nanopore-sispa-da4f2gtn
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: In development
We are still developing and optimizing this protocol
Created: March 22, 2024
Last Modified: March 25, 2024
Protocol Integer ID: 97127
Abstract
Nanopore SISPA
Sample Preprocessing
Sample Preprocessing
Refer to sample preprocessing protocol for appropriate sample type.
Use 280 μl of the filtrate as starting material for the Qiagen Viral RNA Mini Kit.
RNA extraction with QIAamp Viral RNA Mini Kit
RNA extraction with QIAamp Viral RNA Mini Kit
Add 1:500 linear acrylamide instead of carrier RNA to Buffer AVL.
Pipet 1120 μL prepared Buffer AVL into a 2 mL Eppendorf tube.
Add 280 μL of sample into the tube containing Buffer AVL.
Mix by pulse-vortexing for 15 s.
Incubate at room temperature for 10 min.
Briefly centrifuge the tube to remove drops from the inside of the lid.
Add 1120 μL 100% ethanol to the sample, and mix by pulse-vortexing for 15 s.
After mixing, briefly centrifuge the tube to remove drops from inside the lid.
Carefully apply 630 μL of the lysate to the QIAamp Mini column without wetting the rim. Close the cap, and centrifuge at 6000 x g for 1 min.
Place the QIAamp Mini column into a clean 2 mL collection tube, and discard the tube containing the filtrate.
Note
If some lysate does not pass through the membrane after the initial spin, repeat the spin until it has completely flowed through.
Repeat the previous step until all of the lysate has been added to the column.
Carefully open the QIAamp Mini column, and add 500 μL Buffer AW1.
Close the cap, and centrifuge at 6000 x g for 1 min.
Place the QIAamp Mini column in a clean 2 mL collection tube, and discard the tube containing the filtrate.
Carefully open the QIAamp Mini column, and add 500 μL Buffer AW2.
Close the cap and centrifuge at 16,000 x g for 3 min.
Place the QIAamp Mini column in a new 2 mL collection tube, and discard the old collection tube with the filtrate.
Centrifuge at 16,000 x g for 1 min.
Place the QIAamp Mini column in a clean 1.5 mL microcentrifuge tube. Discard the old collection tube containing the filtrate.
Carefully open the QIAamp Mini column and add 50 μL Buffer AVE equilibrated to room temperature.
Close the cap, and incubate at room temperature for 1 min.
Centrifuge at 6000 x g (8000 rpm) for 1 min to collect eluate.
DNase treatment and RNA cocentration
DNase treatment and RNA cocentration
Mix 50 μL eluate with 100 μL of RNA Binding Buffer.
Add and mix in 150 μL of 100% ethanol.
Transfer the sample to the Zymo-Spin IC Column3 in a CollectionTube and centrifuge.
Discard the flow-through
Add 400 μl RNA Wash Buffer to the column, centrifuge and discard the flow-through.
Make up a DNase I Reaction Mix for each sample to be treated:
Component Volume
DNase I 5 µL
DNA Digestion Buffer 35 µL
Add 40 μl of DNase I Reaction Mix directly into column matrix and incubate at room temperature for 15 minutes.
Add 700 μl RNA Wash Buffer to the column and centrifuge. Discard the flow-through.
Add 400 μl RNA Wash Buffer to the column and centrifuge for 1 minute ensure complete removal of the wash buffer.
Carefully, transfer the column into a RNase-free tube.
Add 10 μl DNase/RNase-Free Water directly to the column matrix and centrifuge.
Note
Pause point: store RNA at -20C
FastSelect rRNA removal
FastSelect rRNA removal
Bring FastSelect -rRNA HMR, FastSelect 5S/16S/23S, FastSelect FH Buffer, and Nuclease-free Water to room temperature.
For FastSelect 5S/16S/23S, incubate the tube at 37°C for 5 min, and then vortex to dissolve the precipitate.
Mix all other reagents vigorously by vortexing, and then briefly centrifuge.
Prepare depletion master mix on ice:
Component Volume
FastSelect FH Buffer 1.5 µL
FastSelect -rRNA HMR 1 µL
FastSelect 5S/16S/23S 1 µL
Nuclease-free Water 1.5 µL
Add 5 ul of master mix to template RNA.
Incubation reactions as follows:
1. 2 min at 75 °C
2. 2 min at 70 °C
3. 2 min at65 °C
4. 2 min at60 °C
5. 2 min at 55 °C
6. 2 min at37 °C
7. 2 min at 25 °C
8. Hold at 4°C
Add 19.5 μl QIAseq Beads (prewarmed to room temperature and mixed by vortexing) to the 15 μl reaction.
Mix thoroughly by vortexing, and then incubate for 5 min at room temperature.
Centrifuge in a table-top centrifuge until the beads are completely pelleted (~2 min).
Place the tubes/plate on a magnetic rack for 2 min. Once the solution has cleared, with the beads still on the magnetic stand, carefully remove and discard the supernatant.
Note
Do not discard the beads, because they contain the RNA of interest.
Add 15 μl of Nuclease-free Water and 19.5 μl of QIAseq Bead Binding Buffer (prewarmed to room temperature and mixed by vortexing). Mix vigorously by vortexing, and then incubate for 5 min at room temperature.
Centrifuge in a table-top centrifuge until the beads are completely pelleted (~2 min).
Place the tubes/plate on a magnetic rack for 2 min. Once the solution has cleared, with the beads still on the magnetic stand, carefully remove and discard the supernatant.
With the beads still on the magnetic stand, add 200 μl of 80% ethanol. Wait for 30 s.
Carefully remove and discard the wash.
Note
Do not discard the beads, because they contain the RNA of interest.
Repeat the ethanol wash.
Note
Completely remove all traces of ethanol after this second wash. Remove the ethanol with a 200 μl pipette tip first, and then use a 10 μl pipette tip to remove any residual ethanol that will settle.
With the beads still on the magnetic stand, air-dry at room temperature for 6–10 min until all liquid has evaporated but without overdrying the beads.
Note
Visually inspect the pellet to confirm that it is completely dry.
Remove the beads from the magnetic stand, and elute the RNA from the beads by adding 12 μl Nuclease-free Water. Mix well by pipetting or vortexing, and then allow the tubes to sit at room temperature for 2 min to appropriately hydrate the beads.
Return the tubes/plate to the magnetic rack until the solution has cleared.
Transfer 10 μl of the supernatant to nuclease-free tube.
Note
Pause point: Samples can be store at -80C
Reverse Transcription
Reverse Transcription
Prepare RT reaction master mix. Per sample volumes:
Component Volume
DNase I treated RNA 6 µL
dNTP 10mM 0.5 µL
SISPA-A primer 40uM 1 µL
Nuclease-free water 2.5 µL
Mix and briefly centrifuge.
Incubate at 65C for 5min and then on ice for 1 min
Pre-warm the 5x SSIV buffer to RT. Vortex and briefly centrifuge.
Prepare RT mastermix:
Component Volume
5x SSIV Buffer 2 µL
DTT 100 mM 0.5 µL
RNase Inhibitor 0.5 µL
Reverse Transcriptase 0.5 µL
Add 3.5ul of RT master mix to annealed RNA.
Incubate at 42C for 10 min and then at 80C for 10 min.
Second Strand cDNA Synthesis (Klenow fragment)
Second Strand cDNA Synthesis (Klenow fragment)
Add 1ul of 10x NEB buffer 2 to the RT product.
Incubate at 95C for 3 min and then one ice for 1min.
Add 0.5ul of Klenow fragment to the mixture.
Incubate at 37C for 1 h and then at 75C for 15 min.
AMPure cleanup
AMPure cleanup
Prepare the AMPure XP beads for use; resuspend by vortexing.
Transfer the cDNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
Add 11 μl of resuspended AMPure XP beads to the cDNA samples and mix by pipetting up and down.
Incubate for 5 minutes at room temperature.
Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
Keep on magnet, wash beads with 200 μl of freshly prepared 70% ethanol without disturbing the pellet. Remove the 70% ethanol using a pipette and discard.
Repeat the previous step.
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for 3 min, but do not dry the pellet to the point of cracking.
Remove the tube from the magnetic rack and resuspend pellet in 10 μl nuclease-free water. Incubate for 2 minutes at room temperature.
Pellet the beads on a magnet until the eluate is clear and colourless.
Remove and retain 10 μl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Single Primer PCR amplification
Single Primer PCR amplification
Setup PCR master mix:
Component Volume
2x LongAmp PCR buffer 12.5 µL
SISPA-B primer 10 uM 5 µL
Nuclease-free water 5 µL
Note
Mastermix should be made up and aliquoted into PCR tubes in a mastermix cabinet. Cabinet and tubes should be cleaned with decontamination wipes/70% ethanol and UV sterilised before and after use.
Program the following PCR cycles into the thermal cycler.
Step Temperature Time Cycles
Inital Denaturation 95C 3 min 1
Denaturation 95C 15 s 20
Annealing 50C 30 s 20
Extension 65C 50 s 20
Final Extension 65C 5 min 1
AMPure cleanup 2
AMPure cleanup 2
Prepare the AMPure XP beads for use; resuspend by vortexing.
Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
Add 25 μl of resuspended AMPure XP beads to the PCR product and mix by pipetting up and down.
Incubate for 5 minutes at room temperature.
Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
Keep on magnet, wash beads with 200 μl of freshly prepared 70% ethanol without disturbing the pellet. Remove the 70% ethanol using a pipette and discard.
Repeat the previous step.
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for 3 min, but do not dry the pellet to the point of cracking.
Remove the tube from the magnetic rack and resuspend pellet in 24 μl nuclease-free water. Incubate for 2 minutes at room temperature
Pellet the beads on a magnet until the eluate is clear and colourless.
Remove and retain 24 μl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Note
Pause point: Store samples at -20C
Native barcoding and sequencing by ligation
Native barcoding and sequencing by ligation
Refer to the nanopore protocol.