Apr 17, 2025
FLEXIR-seq - Full-Length targEted capture using eXon probes for IsofoRms
- Lee Murphy1,
- Simon Biddie2,
- Giovanna Weykopf2,
- Audrey AC Coutts1
- 1Genetics Core, Edinburgh Clinical Research Facility, University of Edinburgh;
- 2University of Edinburgh
Protocol Citation: Lee Murphy, Simon Biddie, Giovanna Weykopf, Audrey AC Coutts 2025. FLEXIR-seq - Full-Length targEted capture using eXon probes for IsofoRms. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj9dnwlk5/v1
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: Working
We use this protocol and it's working
Created: December 18, 2024
Last Modified: April 17, 2025
Protocol Integer ID: 115963
Keywords: RNA, isoforms, capture, long-read, nanopore, ONT, Twist, FLEXIR-seq
Abstract
This protocol outlines the procedure for Target enrichment of cDNA using Twist probes for sequencing on ONT PromethION. It is used to investigate full-length transcript isoforms including transcripts that are lowly-expressed, and which are cell-type specific. It provides a powerful method for deep, quantitative, characterisation of transcript isoforms.
Materials
Consumables
Beckman Coulter Agencourt RNAClean XP beads (A63987)
Beckman Coulter Agencourt AMPure XP beads (A63881)
Eppendorf 1.5 ml DNA LoBind tubes (E0030108051)
Invitrogen Dynabeads M-270 Streptavidin (65305)
Life Technologies RNaseOUT 40 U/μl (10777019)
NEB NEBNext Quick Ligation Reaction Buffer (B6058)
NEB T4 DNA Ligase 2M U/ml (M0202M)
NEB Lambda Exonuclease (M0262L)
NEB Thermolabile Exonuclease I (M0568)
NEB USER (Uracil-Specific Excision Reagent) Enzyme (M5505L)
NEB 10 mM dNTP solution (N0447)
NEB LongAmp Hot Start Taq 2X Master Mix (M0533S)
ONT cDNA-PCR Barcoding Kit 24 v14 (SQK-PCB114.24)
ONT Ligation Sequencing Kit v14 (SQK-LSK114)
Sigma KOD Xtreme Hot Start DNA polymerase (PN 71975-3)
ThermoFisher Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (EP0752)
Twist Mechanical Fragmentation Library Preparation Kit 1 (101280)
Twist Library Preperation Kit 2 (100401)
Twist Universal Adapter System (101307)
Twist Standard Hyb and Wash Kit v2 (104445)
Twist Universal Blockers (100856)
Twist Custom Long-Read Panel special request (designed on your capture requirements) (107171)
Equipment
Thermocycler (Eppendorf Mastercycler M50 or similar)
Centrifuge (Sigma 1-14 or similar)
Magnet for tubes (Life Technologies DynaMag-2 or similar)
Magnet for plates (Invitrogen Dynal MPC-96 or similar)
ONT PromethION sequencing platform
Section 1: Make long stranded cDNA from total RNA (using ONT cDNA-PCR Barcoding Kit 24 v14 #SQK-PCB114.24)
Section 1: Make long stranded cDNA from total RNA (using ONT cDNA-PCR Barcoding Kit 24 v14 #SQK-PCB114.24)
For each sample, prepare up to 2000ng of total RNA in 10µl nuclease-free water, in 0.2 ml PCR tubes.
Mix the sample by flicking the tube to avoid unwanted shearing. Spin down briefly in a microfuge.
Prepare the following mix per sample:
| A | B | |
| Reagent | Volume (µl) | |
| RNA | 10 | |
| cDNA RT Adapter (CRTA) | 1 | |
| Annealing Buffer (AB) | 1 | |
| Total Volume | 12 |
Mix gently by flicking the tubes, and spin down. Incubate the reactions in the thermal cycler at 60°C for 5 mins, then cool for 5 minutes at room temperature.
After the incubation, to each of the 0.2 ml PCR tubes containing the RNA sample, add the following (ensure the components are thoroughly mixed by flicking the tube and spin down):
| A | B | |
| Reagent | Volume (µl) | |
| RNA sample (from previous step) | 12 | |
| NEBNext Quick Ligation Reaction Buffer | 3.6 | |
| T4 DNA Ligase 2M U/ml | 1.4 | |
| RNaseOUT | 1 | |
| Total | 18 |
Incubate for 10 minutes at room temperature.
After the 10min incubation, to each of the 0.2 ml PCR tubes, add the following (ensure the components are thoroughly mixed by flicking the tube and spin down):
| A | B | |
| Reagent | Volume (µl) | |
| RNA sample (from previous step) | 18 | |
| Lambda Exonuclease | 1 | |
| NEBnext USER (Uracil-Specific Excision Reagent) | 1 | |
| Total | 20 |
Incubate for 5 minutes at 37°C in the thermal cycler.
Resuspend the RNase-free XP beads by vortexing. Add 36 µl of resuspended RNase-free XP beads to each reaction and mix gently by flicking the tubes.
Incubate for 5 minutes at room temperature. Flick the tubes every 45 sec.
Spin down the samples and pellet on a magnet.
Keep the tubes on the magnet, and pipette off the supernatant.
Keep the tubes on the magnet and wash the beads with 100 µl of Short Fragment Buffer
(SFB) as follows:
a- Wash the beads with 100 µl of Short Fragment Buffer (SFB).
b-Keeping the magnetic rack on the benchtop, rotate the tube by 180°. Wait for the beads to migrate towards the magnet and to form a pellet.
c-Rotate the tube 180° again (back to the starting position), and wait for the beads to pellet again.
d-Without disturbing the pellet, remove the Short Fragment Buffer (SFB) using a pipette and discard. Repeat the previous step. Spin down and place the tubes back on the magnet.
Pipette off any residual buffer.
Briefly allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
Remove the tubes from the magnetic rack and resuspend each pellet in 12 µl of nuclease-free water.
Incubate at room temperature for 10 minutes.
Pellet the beads on a magnet until the eluate is clear and colourless.
Remove and retain 12 µl of eluate into a clean 0.2 ml thin-walled PCR tube per sample.
To each of the 0.2 ml PCR tubes, add the following:
Ensure the components are thoroughly mixed by flicking the tubes and spin down.
| A | B | |
| Reagent | Volume (µl) | |
| Eluted sample (from previous step) | 12 | |
| RT Primer (RTP) | 1 | |
| dNTPs (10 mM) | 1 | |
| Total | 14 |
Incubate the reaction for 5 minutes at room temperature.
To each of the 0.2 ml PCR tubes, add the following:
Ensure the components are thoroughly mixed by flicking the tubes and spin down.
| A | B | |
| Reagent | Volme (µl) | |
| RT primed sample (from previous step) | 14 | |
| Maxima H Minus 5x RT Buffer | 4.5 | |
| RNaseOUT | 1 | |
| Strand Switching Primer II (SSPII) | 2 | |
| Total | 21.5 |
Incubate at 42°C for 2 minutes in the thermal cycler.
Add 1 µl of Maxima H Minus Reverse Transcriptase to each tube. The total volume will be 22.5 µl per tube. Mix gently by flicking the tubes, and spin down.
Incubate using the following protocol using a thermal cycler:
| A | B | C | D | |
| Cycle step | Temperature | Time | No. of cycles | |
| Reverse transcription and strand-switching | 42°C | 30 mins | 1 | |
| Heat inactivation | 85°C | 5 mins | 1 | |
| Hold | 4°C |
The cDNA can be stored at -20C overnight.
Section 2: Twist Long Read Library preparation RNA (using Twist Mechanical Fragmentation Library Preparation Kit #101280 and Twist Universal Adapter System #101307)
Section 2: Twist Long Read Library preparation RNA (using Twist Mechanical Fragmentation Library Preparation Kit #101280 and Twist Universal Adapter System #101307)
Normalise the cDNA to 500ng input. Dilute the sample in Nuclease Free water (NFW) for a total volume if 35µl. Pipette into a 0.2-ml thin-walled PCR striptube and place on ice.
To each of the 0.2 ml PCR tubes, add the following on ice:
| A | B | |
| Reagent | Volume (μl) | |
| 10x ERA Buffer | 5 | |
| ERA Enzyme Mix | 10 | |
| Total | 15 |
Mix the diluted gDNA sample and reagents by flicking with a finger, then pulse-spin to ensure all of the solution is at the bottom of the tube.
Incubate using the following protocol using a thermal cycler: Start the program to pre-chill the thermal cycler
| A | B | |
| Temperature | Time | |
| 4°C | Hold | |
| 20°C | 30 mins | |
| 65°C | 30 mins | |
| 4°C | Hold |
When the thermal cycler program is complete and the sample block has returned to 4°C, remove the samples from the block and place them on ice.
Add 5 μl Universal Adapters into each sample well. Mix gently by pipetting and keep on ice. Prepare a ligation mix in a 1.5-ml microcentrifuge tube on ice as indicated below.
| A | B | |
| Reagent | Volume (μl) | |
| Water (chilled) | 15 | |
| DNA Ligation Buffer | 20 | |
| DNA Ligation Mix | 10 | |
| Total | 45 |
Add 45 μl ligation Mix well by flicking the tube. Cap the tubes and pulse-spin to ensure all solution is at the bottom of the tube.
Incubate the ligation reaction at 20°C for 1 hour in the thermal cycler, IMPORTANT: Turn off the heated lid or set to the minimum temperature
Then move the samples to the bench top. Proceed to the sample purification.
Vortex the pre-equilibrated DNA Purification Beads until well mixed.
Add 80 μl of homogenized DNA Purification Beads to each ligation sample. Mix well by flicking the tubes.
Incubate the samples for 5 minutes at room temperature.
Place the samples on a magnetic plate for 10 minutes.
The DNA Purification Beads form a pellet, leaving a clear supernatant. Without removing the tubes from the magnetic plate, remove and discard the supernatant.
Wash the bead pellet by gently adding 200 μl freshly prepared 80% ethanol (do not disturb the pellet).
Incubate for 1 minute at room temperature, then remove and discard the ethanol.
Repeat the wash once, for a total of two washes, while keeping the samples on the magnetic plate.
Carefully remove all remaining ethanol with a 10-μl pipette, making sure not to disturb the bead pellet.
Air-dry the bead pellet on the magnetic plate for 5 minutes. IMPORTANT: Do not overdry the beads. This may result in lower recovery of DNA.
Remove the tubes from the magnetic plate and add 37 μl of 0.1x TE buffer to each sample.
Mix by flicking until homogenized. Incubate at room temperature for 2 minutes.
Place the tubes on a magnetic plate and let stand for 3 minutes.
Transfer 35 μl of the clear supernatant containing the ligated and indexed libraries to a clean 0.2-ml thin-walled PCR strip-tube making sure not to disturb the bead pellet.
Prepare a PCR mix in a 1.5-ml microcentrifuge tube on ice as indicated below. Mix well by gentle pipetting.
| A | B | |
| Reagent | Volume (μl) | |
| Water (chilled) | 10 | |
| 2X XtremeBuffer | 100 | |
| dNTPs (2mM) | 40 | |
| KOD Xtreme Hot Start DNA Polymerase | 4 | |
| Total | 154 |
Add 10 μl Twist UDI Primers from the provided 96-well plate to each of the ligated libraries and mix well by gentle flicking.
Add 154 μl PCR master mix to each sample. Pulse-spin the sample tube.
Transfer 100 μl of 200 μl reaction to another tube and immediately transfer it to the thermal cycler.
Incubate using the following protocol. Set the temperature of the heated lid to 105°C.
| A | B | C | D | |
| Step | Temperature | Time | Number of cycles | |
| Initialization | 94°C | 2 minutes | 1 | |
| Denaturation | 98°C | 10 seconds | 8 | |
| Annealing | 58.8°C | 30 seconds | ||
| Extension | 68°C | 10 minutes | ||
| Final Extension | 68°C | 10 minutes | 1 | |
| Final hold | 4°C | Hold | - |
Remove the sample from the block when the thermal cycler program is complete.
Vortex the pre-equilibrated DNA Purification Beads until mixed.
Add 50 μl (0.5X) homogenized DNA Purification Beads to the 1st and 2nd PCR reactions. Mix well by flicking.
Incubate the samples for 5 minutes at room temperature.
Place the samples on a magnetic plate for 1 minute.
The DNA Purification Beads form a pellet, leaving a clear supernatant. Without removing the tubes from the magnetic plate, remove and discard the supernatant.
Wash the bead pellet by gently adding 200 μl freshly prepared 80% ethanol (do not disturb the pellet), incubate for 1 minute at room temperature, then remove and discard the ethanol.
Repeat this wash once, for a total of two washes, while keeping the samples on the magnetic plate.
Carefully remove all remaining ethanol with a 10-μl pipette, making sure not to disturb the bead pellet.
Air-dry the bead pellet on the magnetic plate for 5 minutes. Do not overdry the bead pellet.
Remove the plate or tubes from the magnetic plate and add 22 μl water to the 1st PCR reaction. Mix by flicking until homogenized. NOTE: The 2nd PCR reaction will be eluted using the elution from the 1st PCR reaction
Incubate at room temperature for 2 minutes.
Place the tubes on a magnetic plate and let stand for 3 minutes.
Transfer 22 μl of the supernatant to the 2nd PCR reaction and repeat steps 38-39 for the 2nd PCR reaction.
Transfer 20 μl of the clear supernatant containing the amplified, indexed libraries from both PCR reactions to a clean 0.2-ml thin-walled PCR strip-tube, making sure not to disturb the bead pellet.
Do a DNA Broad range Qubit and run on the Fragment analyser with the standard DNA kit to check for the quantity and size of the library fragments.
STOPPING POINT: If not proceeding immediately to a Twist
Target Enrichment System, store the amplified indexed libraries at –20°C
Section 3: Twist Target Enrichment System (using Twist Standard Hyb and Wash Kit v2 with the custom Panel #104446 and Twist Universal Blockers #100578).
Section 3: Twist Target Enrichment System (using Twist Standard Hyb and Wash Kit v2 with the custom Panel #104446 and Twist Universal Blockers #100578).
In order to multiplex 8 libraries, pipette 500ng of each library into a 0.2-ml thin-walled PCR tube. This is done by using the concentration of each amplified, indexed library to calculate the volume (in μl) of each library needed for hybridization. If the amount of library is insufficient, a smaller amount can be use but is may result in decreased library complexity and a low capture yield.
Dry the indexed library pool using a vacuum concentrator using low or no heat. Once the pool is completely dried down, cap the tube and set aside at room temperature.
Heat the Hybridization Mix at 65°C in the heat block for 10 minutes then cool to room temperature on the benchtop for 5 minutes.
Prepare a probe solution in a clean thin-walled PCR 0.2-ml strip-tube. Mix by pipetting up and down 10 times.
| A | B | |
| Reagent | Volume (μl) | |
| Hybridization Mix | 20 | |
| Custom Panel | 4 | |
| Water | 4 | |
| Total | 28 |
Resuspend the dried indexed library pool (from Step 2) by adding the reagents described below. Mix by pipetting up and down 10 times.
| A | B | |
| Reagent | Volume (μl) | |
| Dried Indexed Library Pool | - | |
| Blocker Solution | 5 | |
| Universal Blockers | 7 | |
| Total | 12 |
Heat the probe solution to 95°C for 2 minutes in a thermal cycler with the lid at 105°C, then immediately cool on ice for 5 minutes.
While the probe solution is cooling on ice, heat the tube containing the resuspended indexed library pool at 95°C for 5 minutes in a thermal cycler with the lid at 105°C
Equilibrate both the probe solution and resuspended indexed library pool to room temperature on the benchtop for 5 minutes.
Vortex and spin down the probe solution, then transfer the entire volume to the resuspended indexed library pool. Mix well by vortexing.
Pulse-spin the tube to ensure all solution is at the bottom of the tube.
Add 30 μl Hybridization Enhancer to the top of the entire capture reaction.
Pulse-spin the tube to ensure there are no bubbles present. IMPORTANT: Seal the tube tightly to prevent excess evaporation over the 16-hour incubation.
Incubate the hybridization reaction at 70°C for 16 hours overnight in a thermal cycler with the lid at 85°C. NOTE: Halting hybridization between 15–17 hours will not affect downstream capture quality.
Before starting step 82. Do the following:
- Preheat the following tubes at 48°C until any precipitate is dissolved: Binding Buffer, Standard Wash Buffer 1 and Wash Buffer 2
- For each hybridization reaction:
- Equilibrate 800 µl Binding Buffer to room temperature
- Equilibrate 225 μl Std Wash Buffer 1 to 68ºC
- Leave 700 µl Wash Buffer 2 at 48°C
- Equilibrate the Dynabeads M-270 Streptavidin Beads to room temperature for at least 30 minutes
- Make fresh 0.2 N NaOH using 100 µL of 2 N NaOH into 900 μL of molecular biology grade water
Vortex the pre-equilibrated Streptavidin Binding Beads until mixed.
Add 100 μl Streptavidin Binding Beads to a 1.5-ml microcentrifuge tube. Prepare one tube for each hybridization reaction.
Add 200 μl Binding Buffer to the tube and mix by pipetting.
Place the tube on a magnetic stand for 1 minute, then remove and discard the clear supernatant. Make sure to not disturb the bead pellet. Remove the tube from the magnetic stand
Repeat the wash (Steps 85 and 86) two more times for a total of three washes.
After removing the clear supernatant from the third wash, add a final 200 μl Binding Buffer and resuspend the beads by vortexing until homogenized.
Heat the resuspended beads at 68°C for at least 10 min.
After the hybridization of the library is complete, open the thermal cycler lid and directly transfer the volume of each preheated Streptavidin Binding Beads from Step 89 into the corresponding tube of hybridization reaction. Mix by pipetting and flicking.
IMPORTANT: Rapid transfer directly from the thermal cycler at 70°C is a critical step for minimizing off-target binding. Do not remove the tube of hybridization reaction from the thermal cycler or otherwise allow it to cool to less than 70°C before transferring the solution to the washed Streptavidin Binding Beads. Allowing to cool to room temperature for less than 5 minutes will result in as much as 10–20% increase in off-target binding.
Incubate the tube of the hybridization reaction with the Streptavidin Binding Beads for 5 minutes at 68°C, agitation is not required. NOTE: Do not vortex. Aggressive mixing is not required.
Remove the tube containing the hybridization reaction with Streptavidin Binding Beads from the mixer and pulse-spin to ensure all solution is at the bottom of the tube.
Place the tube on a magnetic stand for 1 minute.
Remove and discard the clear supernatant including the Hybridization Enhancer. Do not disturb the bead pellet.
Remove the tube from the magnetic stand and add 200 μl 68°C Standard Wash Buffer 1. Mix by flicking.
Incubate the tube for 5 minutes at 68°C.
Pulse-spin to ensure all solution is at the bottom of the tube(s) and transfer the entire volume from Step 96 (~200 μl) into a new 1.5-ml microcentrifuge tube, one per hybridization reaction. Place the tube on a magnetic stand for 1 minute.
IMPORTANT: This step reduces background from non-specific binding to the surface of the tube
Remove and discard the clear supernatant. Make sure to not disturb the bead pellet.
Remove the tube from the magnetic stand and add 200 μl of 48°C Wash Buffer 2. Mix by flicking, then pulse-spin to ensure all solution is at the bottom of the tube.
Incubate the tube for 5 minutes at 48°C.
Place the tube on a magnetic stand for 1 minute.
Remove and discard the clear supernatant. Make sure to not disturb the bead pellet.
Repeat the wash (Steps 99-102) two more times, for a total of three washes.
After the final wash, use a 10 μl pipette to remove all traces of supernatant. Proceed immediately to the next step. Do not allow the beads to dry.
Remove the tube from the magnetic stand and add 12 μl water. Mix by flicking until homogenized, then place this solution, hereafter referred to as the Streptavidin Binding Bead slurry, on ice.
Add 12 μl 0.2 N NaOH (freshly diluted from 2 N NaOH) to the Streptavidin Binding Bead slurry. Vortex and spin down. Incubate at room temperature for 5 minutes.
Add 24 μl 200 mM Tris-HCl pH 8 to the mixture from step 37. Vortex and spin down.
Place the mixture on a magnetic rack and transfer the supernatant into a clean thin-walled PCR 0.2-ml strip-tube. Discard the beads.
Prepare a PCR mixture by adding the following reagents to the tube containing the supernatant. Mix by pipetting. Split 200 μl reaction into two 100 μl tubes.
| A | B | |
| Reagent | Volume (μl) | |
| Supernatant from Step 108 | 50 | |
| Amplification Primers | 6 | |
| 2X XtremeBuffer | 100 | |
| dNTPs (2mM ) | 40 | |
| KOD Xtreme Hot Start DNA Polymerase | 4 | |
| Total | 200 |
Pulse-spin the tube, transfer them to the thermal cycler. Incubate using the following protocol. Set the temperature of the heated lid to 105°C.
| A | B | C | D | |
| Step | Temperature | Time | Number of cycles | |
| Initialization | 94°C | 2 mins | 1 | |
| Denaturation | 98°C | 10 secs | 17 | |
| Annealing | 58.8°C | 30 secs | ||
| Extension | 68°C | 10 mins | ||
| Final Extension | 68°C | 10 mins | 1 | |
| Final hold | 4°C | Hold | - |
Vortex the pre-equilibrated DNA Purification Beads until well mixed.
Combine the two separated 100 μl reactions into one 1.5-ml tube.
Add 100 μl (0.5X) DNA Purification Beads to the 1.5-ml tube. Mix well by flicking.
Incubate for 5 minutes at room temperature.
Place the tube on a magnetic plate for 1 minute or until the supernatant is clear.
The DNA Purification Beads form a pellet, leaving a clear supernatant. Without removing the plate or tube from the magnetic plate, remove and discard the clear supernatant.
Wash the bead pellet by gently adding 500 μl freshly prepared 80% ethanol (do not disturb the pellet).
Incubate for 1 minute at room temperature, then remove and discard the ethanol.
Repeat this wash once more, for a total of two washes, while keeping the tube on the magnetic plate.
Carefully remove all remaining ethanol using a 10 μl pipette, making sure to not disturb the bead pellet.
Air-dry the bead pellet on the magnetic plate for 5 minutes or until the bead pellet is dry. Do not overdry the bead pellet.
Remove the tube from the magnetic plate and add 42 μl water to each capture reaction. Mix by flicking until homogenized.
Incubate at room temperature for 2 minutes.
Place the plate or tube on a magnetic plate and let stand for 3 minutes.
Transfer 40 μl of the clear supernatant containing the enriched library to a clean thin-walled PCR 0.2-ml tube making sure to not disturb the bead pellet.
Validate and quantify each enriched library using the Agilent bioanalyser with the High sensitivity assay and a Qubit dsDNA High Sensitivity Assay.
Section 4: ONT Ligation (using ONT Ligation Kit V14 #SQK-LSK114). The aim is to ligate ONT adapters to the enriched libraries for sequencing on the PromethION.
Section 4: ONT Ligation (using ONT Ligation Kit V14 #SQK-LSK114). The aim is to ligate ONT adapters to the enriched libraries for sequencing on the PromethION.
Add 10 ul of NFW to the 40ul of enriched library
Add the following to the 50ul of library. Thoroughly mix the reaction by gently flicking and briefly spinning down
| A | B | |
| Reagent | Volume (µl) | |
| NEBnext Ultra II End-prep Reaction Buffer | 7 | |
| NEBnext Ultra II End-prep Enzyme Mix | 3 | |
| Total | 10 |
Using a thermal cycler, incubate using the following protocol
| A | B | |
| Temperature | Time | |
| 20°C | 5 mins | |
| 65°C | 5mins |
Resuspend the AMPure XP Beads (AXP) by vortexing.
Add 60 µl of resuspended the AMPure XP Beads (AXP) to the end-prep reaction and mix by flicking the tube.
Incubate for 5 minutes at room temperature. Every 1 minute gently flick the tube.
Prepare 500 μl of fresh 80% ethanol in nuclease-free water.
Spin down the sample and pellet on a magnet until supernatant is clear and colourless. Keep the tube on the magnet, and pipette off the supernatant.
Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
Repeat step 135
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for 30 seconds, but do not dry the pellet to the point of cracking.
Remove the tube from the magnetic rack and resuspend the pellet in 61 µl nuclease free water. Incubate for 2 minutes at room temperature.
Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
Remove and retain 61 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Spin down the Ligation Adapter (LA) and T4 DNA Ligase, and place on ice.
Thaw Ligation Buffer (LNB) at room temperature, spin down and mix by pipetting. Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.
Thaw the Elution Buffer (EB) at room temperature and mix by vortexing.
Thaw either Short Fragment Buffer (SFB) at room temperature and mix by vortexing. Then spin down and place on ice.
In a 1.5 ml Eppendorf DNA LoBind tube, mix in the following order. Thoroughly mix the reaction by gently flicking and briefly spinning down.
| A | B | |
| Reagent | Volume (µl) | |
| DNA sample from the previous step | 60 | |
| Ligation Adapter (LA) | 5 | |
| Ligation Buffer (LNB) | 25 | |
| T4 DNA Ligase | 10 | |
| Total | 100 |
Thoroughly mix the reaction by gently flicking and briefly spinning down. Incubate the reaction for 10 minutes at room temperature.
Resuspend the AMPure XP Beads (AXP) by vortexing.
Add 40 µl of resuspended AMPure XP Beads (AXP) to the reaction and mix by flicking the tube.
Incubate for 5 minutes at room temperature. Every 1 minute gently flick the tube.
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
Wash the beads by adding either 250 μl Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.
Repeat step 151.
Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for 30 seconds, but do not dry the pellet to the point of cracking.
Remove the tube from the magnetic rack and resuspend the pellet in 25 µl Elution Buffer (EB).
Spin down and incubate for 10 minutes at room temperature.
Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
Remove and retain 32 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
The prepared library is used for loading into the PromethION flow cell. Store the library ice or at 4°C until ready to load. 50 fmol is required for loading onto the flow cell.
Quantify the library using the Qubit DNA high sensitivity assay.
Protocol references
Oxford Nanopore Technologies cDNA-PCR Barcoding Kit 24 V14 protocol (pcr-cdna-sequencing-v14-barcoding-sqk-pcb114-24-PCB_9201_v114_revC_06Dec2023-minion)
Oxford Nanopore Technologies Ligation sequencing amplicons V14 (SQK-LSK114 (VACDE_9163_v114_revU_29Jun2022)
Twist Long Read Library Preparation and Standard Hyb v2 Enrichment (DOC-001320 REV 2.0)