Apr 20, 2022
Version 2
DNA extraction and Nanopore library prep from single wild-caught drosophilids V.2
Version 1 is forked from DNA extraction and Nanopore library prep from 15-30 whole flies
- 1Petrov Lab, Stanford University
Protocol Citation: Bernard Y Kim 2022. DNA extraction and Nanopore library prep from single wild-caught drosophilids. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpbdn8lzp/v2Version created by Bernard Y Kim
Manuscript citation:
Kim, B. Y., Wang, J. R., Miller, D. E., Barmina, O., Delaney, E., Thompson, A., ... & Petrov, D. A. (2021). Highly contiguous assemblies of 101 drosophilid genomes. Elife, 10, e66405. https://doi.org/10.7554/eLife.66405
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: April 20, 2022
Last Modified: April 20, 2022
Protocol Integer ID: 61103
Keywords: Drosophila, nanopore, ligation, HMW, single fly
Abstract
We have been assembling the genomes of many Drosophila species. This protocol fork allows for the sequencing of single wild-caught flies using Nanopore MinION sequencers, in contrast to our previous protocol that originally used gDNA extracted from tens or hundreds of flies from an inbred line.
While read lengths are not nearly close to what we were seeing before, libraries range from 2-10kb read N50 and typically produce genome assemblies with contig N50 >1 Mbp.
The protocol for ultra-long read sequencing from large pools of flies can be found here: dx.doi.org/10.17504/protocols.io.bdfqi3mw
For now, please cite the original genomes paper if you use this protocol: https://doi.org/10.7554/eLife.66405
An update that includes many single wild-caught fly genomes is in progress.
Guidelines
Flies were collected from the wild and stored in 95% ethanol at -20C. We have successfully assembled genomes from samples ranging from 4 months to 15 years old. Younger samples perform better, of course. Ideally the samples are no older than 5 years and stored properly (e.g. alcohol has not evaporated).
Apart from the gDNA extraction steps, this protocol mostly follows the standard LSK110 protocol. Slight modifications were made to improve library recovery and pore occupancy.
Materials
MATERIALS
10% SDS solution
NEBNext Companion Module forOxford Nanopore Technologies Ligation Sequencing – 24 rxnsNew England BiolabsCatalog #E7180S
Oxford Nanopore Ligation Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-LSK110
ChloroformMillipore SigmaCatalog #CX1055-6
Phenol Chloroform Isoamyl Alcohol (25:24:1) Tris-saturated (pH 8.0)Fisher ScientificCatalog #BP1752I-400
3M sodium acetate
Proteinase K Solution (20 mg/mL) RNA gradeThermo Fisher ScientificCatalog #25530049
Monarch RNase A – 1 ml (2x0.5ml)New England BiolabsCatalog #T3018L
Homogenization Buffer (HB) [0.1M NaCl 30mM Tris-HCl pH 8.0 10 mM EDTA 0.5% Triton X-100]
Lysis Buffer (LB) [0.1M Tris-HCl pH 8.0; 0.1M NaCl; 20mM EDTA]
Hydration Buffer (STE) [400mM NaCl 20mM Tris-HCl pH 8.0 30mM EDTA]
DNAse wash buffer (DWB) [300mM KCl 2mM CaCl2 10mM MgCl2 15 mM HEPES pH 8.0]
Elution Buffer (EB) [10 mM Tris-HCl pH 8.0]
DNA extractions are performed in Phase lock gel tubes to minimize handling and to maximize yield. A cheaper alternative to the official phase lock gel tubes is to put ~200uL of Dow Corning High Vacuum Grease into a 2.0 mL LoBind tube with a small syringe. Care should be take with homebrew phase lock gel tubes as using too little grease will result in the phase lock layer collapsing during the chloroform extraction step.
Equipment
DNA LoBind tubes, 1.5 mL
NAME
Tubes
TYPE
Eppendorf
BRAND
022431021
SKU
LINK
1.5 mL
SPECIFICATIONS
Equipment
DNA LoBind tubes, 1.5 mL
NAME
Tubes
TYPE
Eppendorf
BRAND
022431048
SKU
LINK
1.5 mL
SPECIFICATIONS
Equipment
Large-orifice pipet tips, 200uL
NAME
Pipette tips
TYPE
Fisher
BRAND
02-707-134
SKU
LINK
200 uL
SPECIFICATIONS
Equipment
Dounce Homogenizer, 2mL
NAME
Tissue Grinder
TYPE
Kimble
BRAND
885300-0002
SKU
LINK
2 mL with Pestles A and B
SPECIFICATIONS
Equipment
5PRIME Phase Lock Gel tube, light, 2mL
NAME
Quantabio
BRAND
2302820
SKU
LINK
Light
SPECIFICATIONS
Protocol materials
Lysis Buffer (LB) [0.1M Tris-HCl pH 8.0; 0.1M NaCl; 20mM EDTA]
10% SDS solution
Elution Buffer (EB) [10 mM Tris-HCl pH 8.0]
3M sodium acetate
Homogenization Buffer (HB) [0.1M NaCl 30mM Tris-HCl pH 8.0 10 mM EDTA 0.5% Triton X-100]
NEBNext Companion Module forOxford Nanopore Technologies Ligation Sequencing – 24 rxnsNew England BiolabsCatalog #E7180S
ChloroformMerck MilliporeSigma (Sigma-Aldrich)Catalog #CX1055-6
Proteinase K Solution (20 mg/mL) RNA gradeThermo Fisher ScientificCatalog #25530049
Hydration Buffer (STE) [400mM NaCl 20mM Tris-HCl pH 8.0 30mM EDTA]
DNAse wash buffer (DWB) [300mM KCl 2mM CaCl2 10mM MgCl2 15 mM HEPES pH 8.0]
Monarch RNase A – 1 ml (2x0.5ml)New England BiolabsCatalog #T3018L
Oxford Nanopore Ligation Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-LSK110
Phenol Chloroform Isoamyl Alcohol (25:24:1) Tris-saturated (pH 8.0)Fisher ScientificCatalog #BP1752I-400
DNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
Hydration Buffer (STE) [400mM NaCl 20mM Tris-HCl pH 8.0 30mM EDTA]
Hydration Buffer (STE) [400mM NaCl 20mM Tris-HCl pH 8.0 30mM EDTA]
Lysis Buffer (LB) [0.1M Tris-HCl pH 8.0; 0.1M NaCl; 20mM EDTA]
DNA LoBind Tubes 2.0 mLEppendorfCatalog #30108078
Phenol Chloroform Isoamyl Alcohol (25:24:1) Tris-saturated (pH 8.0)Fisher ScientificCatalog #BP1752I-400
ChloroformMerck MilliporeSigma (Sigma-Aldrich)Catalog #CX1055-6
DNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
3M sodium acetate
Elution Buffer (EB) [10 mM Tris-HCl pH 8.0]
NEBNext Companion Module forOxford Nanopore Technologies Ligation Sequencing – 24 rxnsNew England BiolabsCatalog #E7180S
PCR Tubes, 0.2mL, flat cap, natural, PCR Tube; 0.2mL; Natural; w/flat cap; 1000/Pk.Thermo FisherCatalog #3412
Nuclease-free water or water filtered using a Milli-Q filtering systemAmbionCatalog #AM9932
DNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
Large-Orifice Pipet Tips 200μLFisher ScientificCatalog #02-707-134
Agencourt AMPure XPBeckman CoulterCatalog #A63880
DNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
NEBNext Companion Module forOxford Nanopore Technologies Ligation Sequencing – 24 rxnsNew England BiolabsCatalog #E7180S
Oxford Nanopore Ligation Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-LSK110
DNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
NEBNext Companion Module forOxford Nanopore Technologies Ligation Sequencing – 24 rxnsNew England BiolabsCatalog #E7180S
Ligation sequencing kit 1DOxford Nanopore TechnologiesCatalog #SQK-LSK109
Oxford Nanopore Ligation Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-LSK110
Large-Orifice Pipet Tips 200μLFisher ScientificCatalog #02-707-134
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Oxford Nanopore Ligation Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-LSK110
Elution Buffer (EB) [10 mM Tris-HCl pH 8.0]
DNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
Oxford Nanopore Ligation Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-LSK110
DNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
Oxford Nanopore Ligation Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-LSK110
Oxford Nanopore Ligation Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-LSK110
Large-Orifice Pipet Tips 200μLFisher ScientificCatalog #02-707-134
Flowcell Wash KitOxford Nanopore TechnologiesCatalog #EXP-WSH003
Monarch RNase A – 1 ml (2x0.5ml)New England BiolabsCatalog #T3018L
Proteinase K Solution (20 mg/mL) RNA gradeThermo ScientificCatalog #25530049
10% SDS solution
(Optional) Hydration of ethanol-fixed tissue
(Optional) Hydration of ethanol-fixed tissue
45m
45m
Place flies on a sheet of filter paper and briefly dab with a Kimwipe to remove excess ethanol, then transfer the fly to a 1.5 mL LoBind tube.
DNA LoBind Tubes, 1.5 mLSigma AldrichCatalog #0030108051
Add 500 µL Buffer STE to the tube with the fly.
Hydration Buffer (STE) [400mM NaCl 20mM Tris-HCl pH 8.0 30mM EDTA]Sigma Aldrich
Incubate on a platform rocker or rotator for 00:15:00 at low to medium speed.
15m
Replace the solution with 500 µL o of fresh Buffer STE and incubate for another 00:15:00 on the rocker at low to medium speed.
Hydration Buffer (STE) [400mM NaCl 20mM Tris-HCl pH 8.0 30mM EDTA]Sigma Aldrich
15m
Use a pipette to remove as much of the liquid as possible, but do not dry the fly.
Tissue homogenization
Tissue homogenization
5m
5m
Add 283 µL lysis buffer to the tube with the fly.
Lysis Buffer (LB) [0.1M Tris-HCl pH 8.0; 0.1M NaCl; 20mM EDTA]Sigma Aldrich
Using a pellet pestle, manually homogenize the fly tissue. While it's important to squish the fly enough so that soft tissue can be digested, it is okay for some bigger pieces of tissue, particularly those held together by tough cuticle like the legs, to remain.
Equipment
Pellet Pestle
NAME
Kimble Kontes
BRAND
K749520-1590
SKU
LINK
1.5 mL Polypropylene w/o Microtubes RNAse DNase free
SPECIFICATIONS
Note
TIP: Make sure fly bits aren't stuck on the pestle before discarding. Every bit of tissue counts!
Tissue lysis
Tissue lysis
Add 7.5 µL 20 mg/mL Proteinase K, 7.5 µL 10% SDS, and 2 µL 20 mg/mL RNAse A to the tube. Gently flick and swirl to mix.
Monarch RNase A – 1 ml (2x0.5ml)Sigma AldrichCatalog #T3018L
Proteinase K Solution (20 mg/mL) RNA gradeSigma AldrichCatalog #25530049
10% SDS solutionSigma Aldrich
Incubate tube for 02:00:00 to 04:00:00 at 50 °C . Gently mix tube every 00:45:00 .
Note
The tube should go from cloudy (due to undigested tissue bits) to clear as the digestion proceeds. Due to the pestle homogenization there will usually be bigger pieces of the fly, like the wings and legs, floating around. This is fine, just pipette as much liquid as you can get for the next step.
4h
Phenol chloroform extraction
Phenol chloroform extraction
1h
1h
Spin down 1 2mL phase lock gel tube per sample at 15000 x g for 00:00:30 .
Note
Although not essental, phase lock gel tubes help minimize shearing and loss of yield caused by repeated pipetting. Dow Corning High Vacuum Grease is compositionally identical to the light phase lock gel material. We buy the 5.3oz tube from Amazon and squeeze some into a 10mL BD syringe for dispensing. This size of tube/syringe fits well for minimal mess and hassle. Avoid overfilling and air bubbles. We autoclave but be warned this may cause a mess, so wrap the syringe in foil beforehand.
About 250 µL of grease is placed into a 2mL LoBind tube to make the homebrew phase lock gel tube.
IMPORTANT: If an insufficient amount of grease is applied, the phase lock layer will collapse during the chloroform extraction.
Safety information
WARNING: If you are using normal tubes in lieu of LoBinds, do not use polystyrene tubes for the phenol-chloroform extraction. They will melt and burst in the centrifuge. Polypropylene tubes do not melt.
Equipment
5PRIME Phase Lock Gel tube, light, 2mL
NAME
Quantabio
BRAND
2302820
SKU
LINK
Light
SPECIFICATIONS
DNA LoBind Tubes 2.0 mLSigma AldrichCatalog #30108078
Briefly spin down the homogenate/lysis solution, then transfer to the phase lock gel tube by decanting or pipetting with a wide-bore tip.
Add an equal volume (about 350 µL ) of Tris-saturated phenol chloroform isoamyl alcohol (PCI) to the phase lock tube.
Safety information
This should be performed inside the fume hood.
Phenol Chloroform Isoamyl Alcohol (25:24:1) Tris-saturated (pH 8.0)Sigma AldrichCatalog #BP1752I-400
Mix by placing tubes on a rocker at medium speed for 00:08:00 .
Note
We use a rocking platform, so the tubes are placed on their sides horizontally to maximize the surface area. When solution is well mixed, aqueous (top) layer will be a cloudy milky color.
Centrifuge the phase lock tube at 16000 x g for 00:08:00 . Phase lock layer should now separate aqueous and organic layers.
Perform one more PCI extraction: go to step #12 .
Note
Use the same tube for the entire extraction, i.e. just add another 350uL of PCI/CHCl3 to the tube and proceed. The phase lock layer should hold through the extraction steps.
Add an equal volume (usually 350 µL ) of chloroform to the tube.
Safety information
This should be performed inside the fume hood.
ChloroformSigma AldrichCatalog #CX1055-6
Mix by placing tubes on a rocker at medium speed for 00:08:00 .
Centrifuge the phase lock tube at 16000 x g for 00:08:00 . Phase lock layer should now separate aqueous and organic layers.
Quickly decant the aqueous (top) layer into a fresh 1.5 mL LoBind tube.
Note
We find decanting more effective than pipetting. If the pipette tip touches the phase lock gel, it makes a huge mess.
Try to perform the decanting step in a few seconds, and don't tap/shake the phase lock tube to get the last drops out. Care must be taken as the chloroform weakens the phase lock gel layer. If the phase lock tube is inverted for too long during decanting, the layer will collapse and everything will pour out. It's best to leave a couple of drops behind to avoid the hassle of cleaning this up.
IMPORTANT: It is highly recommended to use LoBind tubes in this and subsequent steps. The coating will prevent DNA sticking to the tube. This is helpful for maximizing yield and minimizing shearing.
Safety information
This should be performed inside the fume hood.
DNA LoBind Tubes, 1.5 mLSigma AldrichCatalog #0030108051
DNA precipitation, wash, and resuspension
DNA precipitation, wash, and resuspension
30m
30m
Chill 100% ethanol on ice and make 500 µL of fresh 70% ethanol using nuclease-free water.
Add 0.1 volume (typically 30 µL ) of 3M sodium acetate to the sample. Gently swirl to mix.
3M sodium acetateSigma Aldrich
Add 2-2.5 volumes (typically 675 µL ) of cold 100% ethanol to the tube, and mix with careful swirling and gentle rocking. It will be difficult to see precipitated DNA, but extractions from larger flies will usually have a few visible strands.
Note
If the extraction tube turns cloudy, it is likely salt precipitation because the solution is too nonpolar. Add water dropwise with thorough mixing and the solution should clear up.
Centrifuge the tube at 5000 x g for 00:05:00 to pellet the DNA.
Note
TIP: the pellet will often be invisible. Centrifuge the tubes with the hinge placed consistently towards the outside of the rotor so that you know where the pellet will be.
5m
While being careful not to disturb the pellet, pipette off the ethanol.
Add 200 µL of 70% ethanol to wash the DNA. Gently swirl to mix.
Centrifuge the tube at 5000 x g for 00:02:00 .
While being careful not to disturb the pellet, pipette off the ethanol.
Note
I usually spin the tube down briefly (~2 sec) and use a P10/P20 to grab the last bit of ethanol in the tube.
Wash the pellet once more: go to step #25 .
Allow the tube to air dry no longer than 00:01:00 . Do not over-dry.
1m
Resuspend in 42 µL 10mM Tris and incubate at 50 °C for 00:30:00 .
Note
Carefully pipette the tris up and down the back of the tube to ensure you've resuspended all the DNA. Sometimes it will be spread out a bit along the back of the tube, rather than pelleted cleanly on the bottom.
Elution Buffer (EB) [10 mM Tris-HCl pH 8.0]Sigma Aldrich
30m
Briefly spin down tube to gather any condensation and store at 4 °C .
DNA resuspension
DNA resuspension
15m
15m
Pipette mix slowly 5X with a P200 tip to ensure proper resuspension.
Check sample concentration and quality of 1 µL aliquots using Qubit and Nanodrop.
Note
Ideally, you'll have recovered >250ng DNA and absorbance ratios 260/280 >1.8 and 260/230 >2.0. We have successfully prepared Nanopore libraries with as little as 208ng of gDNA at this point.
Reserve DNA for short-read sequencing
Reserve DNA for short-read sequencing
15m
15m
Reserve at least 20 ng gDNA in 6 µL Tris for Illumina library prep. Store at -20 °C until ready for library prep.
Note
This is a conservative amount. As of April 2022, we use the Illumina kit with 1/5 reaction volumes to prepare libraries. We have successfully prepared Drosophila Illumina libraries with as little as 4-6 ng of gDNA depending on the kit and protocol. Your mileage may vary.
Note
Although Illumina sequencing may no longer be necessary for genome assembly with the release of new flow cell chemistries, a short-read library will be useful for variant calling if this a wild-caught individual, so this is recommended irrespective of flow cell/kit chemistry.
DNA repair and end-prep
DNA repair and end-prep
Thaw NEBNext repair and dA-tailing mixes and buffers from the Nanopore Companion Module. Mix buffers by vortexing and enzyme mixes by flicking. Spin down tubes and keep chilled on ice.
NEBNext Companion Module forOxford Nanopore Technologies Ligation Sequencing – 24 rxnsSigma AldrichCatalog #E7180S
Add all non-reserved DNA (up to 40 µL ) to a PCR tube. Dilute the HMW DNA with water to a final volume of 48.5 µL . Add 3.5 µL of FFPE DNA repair buffer, 3.5 µL of end-prep reaction buffer, 2 µL of FFPE DNA repair mix, and 3 µL of end-prep reaction mix to the tube. Mix tube with gentle flicking (or very gentle pipetting with a cut-off P200 tip).
PCR Tubes, 0.2mL, flat cap, natural, PCR Tube; 0.2mL; Natural; w/flat cap; 1000/Pk.Sigma AldrichCatalog #3412
Nuclease-free water or water filtered using a Milli-Q filtering systemSigma AldrichCatalog #AM9932
In a thermal cycler, incubate at 20 °C for 01:00:00 then 65 °C for 00:30:00 . After this, sample can be held at 4 °C temporarily until ready to proceed.
Using a cut-off P200 tip (a wide bore will be too small to fit in the PCR tube), gently transfer sample to a 1.5 mL DNA LoBind tube. Add 60 µL AMPure beads. Using a wide-bore P200 tip, quickly but gently mix the tube.
DNA LoBind Tubes, 1.5 mLSigma AldrichCatalog #0030108051
Large-Orifice Pipet Tips 200μLSigma AldrichCatalog #02-707-134
Agencourt AMPure XPSigma AldrichCatalog #A63880
Allow the sample to sit at least 00:05:00 at Room temperature to allow the beads to bind the DNA. Meanwhile, prepare 500 µL fresh 70% ethanol with nuclease-free water.
5m
Pellet the beads by placing the tube onto a magnetic tube rack for 00:05:00 or until sample has completely cleared.
Equipment
Magnetic 1.5 mL tube rack
NAME
Any
BRAND
NA
SKU
5m
Pipette off the supernatant, taking care not to disturb the bead pellet.
Add 175 µL of 70% ethanol. Pipette slowly, with the tip touching the front wall of the tube, so that the pellet is not disturbed.
Pipette off the supernatant, taking care not to disturb the DNA pellet.
Wash the pellet once more with 70% ethanol go to step #42 .
Briefly spin the sample tube down and place back onto the magnet. Remove any remaining drops on the bottom of the tube with a P10.
Equipment
Magnetic 1.5 mL tube rack
NAME
Any
BRAND
NA
SKU
Immediately resuspend bead pellet in 31 µL nuclease-free water.
Incubate the tube on the heat block at 50 °C for 00:30:00 . Every 5 minutes, gently flick the tube to encourage any settled beads to resuspend.
30m
Briefly spin down the tube to collect condensation then place tube back on the magnetic rack. Allow tube to sit for
00:05:00 or until sample has completely cleared.
5m
With a cut-off P200 tip, transfer 31 µL eluate to a fresh 1.5 mL LoBind tube.
DNA LoBind Tubes, 1.5 mLSigma AldrichCatalog #0030108051
Check for recovery with Qubit using 1 µL of sample. Recovery should be at least 150 ng DNA at this point.
Adapter ligation
Adapter ligation
Thaw AMXII, T4 ligase, LNB, and LFB from the NEBNext Nanopore Companion Module and the Nanopore LSK110 kit. Mix AMXII, T4 ligase, and LFB by flicking. Mix LNB by pipetting. Briefly spin the tubes down and keep chilled on ice.
NEBNext Companion Module forOxford Nanopore Technologies Ligation Sequencing – 24 rxnsSigma AldrichCatalog #E7180S
Oxford Nanopore Ligation Sequencing KitSigma AldrichCatalog #SQK-LSK110
Add 30 µL prepared DNA sample, 2.5 µL AMX, and 5 µL T4 ligase to a fresh 1.5 mL DNA LoBind tube. Gently flick the tube to mix.
Note
These are 1/2 the standard prep volumes, hence elution in 31uL in Step 47.
DNA LoBind Tubes, 1.5 mLSigma AldrichCatalog #0030108051
NEBNext Companion Module forOxford Nanopore Technologies Ligation Sequencing – 24 rxnsSigma AldrichCatalog #E7180S
Ligation sequencing kit 1DSigma AldrichCatalog #SQK-LSK109
Add 12.5 µL LNB to the sample. Working quickly, mix by gentle pipetting with a wide-bore tip. DNA precipitation is normal, but if the DNA precipitates before you finish mixing it will stick to your pipette tip and you will lose a significant amount of library.
Oxford Nanopore Ligation Sequencing KitSigma AldrichCatalog #SQK-LSK110
Large-Orifice Pipet Tips 200μLSigma AldrichCatalog #02-707-134
Incubate the reaction mixture at Room temperature for 00:30:00 .
30m
Add 20 µL AMPure beads and mix gently by flicking until uniform in color. Let sample sit for at least 00:05:00 , with occasional gentle mixing if necessary, so the DNA can bind to the beads.
Agencourt AMPure XPSigma AldrichCatalog #A63880
5m
Pellet the beads by placing the tube onto a magnetic tube rack for 00:05:00 or until sample has completely cleared.
Equipment
Magnetic 1.5 mL tube rack
NAME
Any
BRAND
NA
SKU
5m
Pipette off the supernatant, being careful not to disturb the bead pellet.
Remove tube from magnetic rack and add 100 µL of LFB to the tube, flicking to mix.
Oxford Nanopore Ligation Sequencing KitSigma AldrichCatalog #SQK-LSK110
Safety information
DO NOT USE ETHANOL TO WASH PREPARED LIBRARY. It will denature the motor protein.
Pellet the beads by replacing the tube onto the magnetic tube rack for 00:05:00 or until sample has completely cleared.
Equipment
Magnetic 1.5 mL tube rack
NAME
Any
BRAND
NA
SKU
5m
Being careful not to disturb the pellet, pipette off all the supernatant.
Remove the tube from the magnet and briefly spin down. Replace tube onto magnet and remove any remaining drops of supernatant with a P10.
Wash the pellet with LFB one more time. go to step #58
Resuspend beads in 26 µL EB.
Elution Buffer (EB) [10 mM Tris-HCl pH 8.0]Sigma Aldrich
Incubate beads on the heat block at 37 °C for 00:15:00 . Every 5 minutes, gently flick the tube to resuspend any settled beads.
15m
Briefly spin down the tube to collect condensation.
Place sample tube onto magnetic rack for at least 00:05:00 or until sample has cleared.
Equipment
Magnetic 1.5 mL tube rack
NAME
Any
BRAND
NA
SKU
5m
Using a cut-off P200 tip, transfer 26 µL eluate into a fresh 1.5 mL LoBind tube.
DNA LoBind Tubes, 1.5 mLSigma AldrichCatalog #0030108051
Quantify library concentration with Qubit using 1 µL of the prepared library.
Tips for sequencing the library
Tips for sequencing the library
Thaw 1 tube sequencing buffer SBII (SQK-LSK110), 1 tube loading solution LS (SQK-LSK110), 1 tube flush buffer FB (EXP-FLP002), and 1 tube flush tether FLT (EXP-FLP002). Mix SBII, LS, and FB by flicking. Mix FLT with a pipette. Spin down and keep reagents on ice until ready to sequence.
Safety information
Make sure the right versions of reagents from the ligation and/or auxiliary/expansion kits are being used.
Oxford Nanopore Ligation Sequencing KitSigma AldrichCatalog #SQK-LSK110
With a cut off P200 tip, transfer at least 100 ng of prepared library to a fresh 1.5mL LoBind tube. This should not exceed 25 µL in volume (i.e. the volume of the eluate from Step 64).
Note
These libraries tend to be pretty fragmented, with read N50s ranging from 2-10kbp. More than 100 ng library is needed for longer N50 libraries, but it often helps to have 2 library loads so that a flush & reload can be used to improve throughput. So this part is a bit of a guessing game that depends on library yield and sample quality.
DNA LoBind Tubes, 1.5 mLSigma AldrichCatalog #0030108051
Add the volume of LS that will result in a total volume of 25 µL in the LoBind tube. For example, if 15 µL of prepared library was initially added to the tube, add 10 µL of LS for a total 25 µL library + LS.
Oxford Nanopore Ligation Sequencing KitSigma AldrichCatalog #SQK-LSK110
Add 25 µL SBII to the tube for a total volume of 50 µL .
Note
50 uL is significantly lower than the standard preparation of 75 uL library+SQB+LS/LBII. We use this amount because 50 uL is enough to barely cover the flow cell membrane; this is more visibly apparent if using LBII instead of LS. This allows us to improve the concentration of library for very low yield samples.
THIS VOLUME HAS NOT BEEN TESTED ON PROMETHION FLOW CELLS. Validation is needed.
Oxford Nanopore Ligation Sequencing KitSigma AldrichCatalog #SQK-LSK110
Follow the official instructions to prime the flow cell, then add the prepared library to the flow cell. When loading the library, be sure to use a wide-bore pipette tip. Gently pipette mix the library before loading to ensure even distribution of the library across the flow cell membrane.
Large-Orifice Pipet Tips 200μLSigma AldrichCatalog #02-707-134
Over the course of a sequencing run, pores will get clogged and become inactive. If you have enough library, we recommend to flush the flow cell at 10-14 hour intervals to make these pores available again.
Flowcell Wash KitSigma AldrichCatalog #EXP-WSH003