TITLE: DNA extraction and Nanopore library prep from 15-30 whole flies
AUTHORS: Bernard Kim, Danny E. Miller, Jeremy Wang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>We have been assembling the genomes of many </span><span style = "font-style:italic;">Drosophila</span><span> species. With that in mind, we developed this protocol to keep the cost of sequencing down to <$500 per assembly while maintaining a decent number of very long reads. Using these guidelines, a typical Drosophila Nanopore sequencing run should have read N50 of 20-40kbp with 5-15% of data in reads >100kbp. Sequencing is halted at about 40-50X depth of coverage (8-10 Gbp for most species). This of course depends on the quality of the sample, quality and quantity of the prepared library, and the frequency at which the flow cell is flushed and reloaded. We typically run 3-4 species per 2 flow cells, usually for ~14-18 Gbp of data per flow cell. </span></div><br/><div class = "text-block">This protocol borrows several elements from John Tyson's "Rocky Mountain" protocol and we thank him for several insightful discussions. </div><div class = "text-block"><a href="https://www.protocols.io/view/rocky-mountain-adventures-in-genomic-dna-sample-pr-7euhjew" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/rocky-mountain-adventures-in-genomic-dna-sample-pr-7euhjew</span></a></div></div>

[STEPS]
?. [(Optional) Hydration of ethanol-fixed tissue]
Place flies on a sheet of filter paper and briefly dab with a Kimwipe to remove excess ethanol, then transfer the flies to a 1.5 mL tube.
?. [(Optional) Hydration of ethanol-fixed tissue]
Add  Buffer STE to the tube with the flies.
1 mL
?. [(Optional) Hydration of ethanol-fixed tissue]
Incubate on a platform rocker or rotator for   at low to medium speed.
?. [(Optional) Hydration of ethanol-fixed tissue]
Replace the solution with  of fresh Buffer STE and incubate for another   on the rocker at low to medium speed.
1 mL
?. [(Optional) Hydration of ethanol-fixed tissue]
Transfer the tissue to a new sheet of filter paper and wick off excess liquid.
?. [Tissue homogenization]
Chill Dounce homogenizer, homogenization buffer (HB), and lysis buffer (LB) on ice.
?. [Tissue homogenization]
Place flies into Dounce homogenizer with  of HB. Dounce 10 times with looser fitting pestle A, then 10 times with tighter fitting pestle B.
1 mL
WARNING: It is easy to break the glass homogenizer if used improperly. Wrap the base of the homogenizer in a bit of paper towel in case of breakage, then brace it against your hand or another soft surface. Do not brace it against a hard surface like a lab bench. To homogenize, slowly move pestle up and down vertically, without twisting or trying force the pestle down if it feels stuck. When the sample is fully homogenized, you should be able to dounce with minimal resistance from fly chunks. Suction from douncing will help mix the pellet that initially gathers at the bottom.Move the pestly slowly to minimize foaming.
?. [Tissue homogenization]
Use a P200 with a wide-bore tip to transfer the homogenate into a new 1.5 mL tube.
?. [Tissue homogenization]
Rinse the homogenizer with  of HB, and use a wide-bore tip to transfer to the tube with the rest of the homogenate.
500 µl
?. [Tissue homogenization]
Pellet fly material at  for  , preferably at  .
Centrifuge: 2000 34
4 °C
?. [Tissue homogenization]
After homogenate has been pelleted, carefully discard the supernatant by decanting. While still inverted, wipe up white lipid smear near lip of tube with a Kimwipe so that it doesn't a chance to drip down onto the pellet.
?. [Tissue homogenization]
Resuspend pellet in 100uL of HB using vigorous mixing with a wide-bore tip. Transfer homogenate into tube with LB and again mix thoroughly with a wide-bore tip.
?. [Tissue homogenization]
Meanwhile, prepare a solution of  LB,  Proteinase K,  10% SDS, and  RNase A in a 1.5 mL LoBind tube.
380 µl
10 µl
10 µl
2 µl
?. [Lysis]
Incubate lysis tube at  for  to . Mix the tube with gentle rocking and inversion, until solution appears relatively homogeneous, at  intervals.
50 °C
Sometimes a bit of vigorous shaking is needed, especially if there is a lot of materal. This is OK - the end product needs to be sheared for library prep anyway. Qualitative observations suggest that thorough mixing improves DNA yield and purity substantially.
Incubation times as short as 1 hour at 55C have worked. We have not carefully tested how this affects yield.
?. [Phenol chloroform extraction]
Spin down 2 phase lock gel tubes per sample at  for  .
Centrifuge: 15000 34
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 minimial mess and hassle. Avoid overfilling and air bubbles. Autoclave but be warned this may cause a mess, so wrap the syringe in foil beforehand.About   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.Reference: https://bitesizebio.com/18944/diy-phase-separating-gel-clean-and-cheap/
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.
?. [Phenol chloroform extraction]
Transfer the homogenate/lysis solution to the phase lock gel tube by pipetting with a wide-bore tip.
?. [Phenol chloroform extraction]
Add an equal volume (about ) of Tris-saturated phenol chloroform isoamyl alcohol (PCI) to the phase lock tube.
600 µl
This should be performed inside the fume hood.
?. [Phenol chloroform extraction]
Mix by placing tubes on a rocker at medium speed for  .
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.
?. [Phenol chloroform extraction]
Centrifuge the phase lock tube at  for . Phase lock layer should now separate aqueous and organic layers.
Centrifuge: 16000 34
?. [Phenol chloroform extraction]
Perform one more PCI extraction:  .
Use the same tube for the second extraction, i.e. just add another 600uL of PCI to the tube and proceed. The phase lock layer should hold through the extraction steps.
?. [Phenol chloroform extraction]
Decant aqueous (top) layer into a fresh phase lock tube. Add an equal volume (usually ) of chloroform to the tube.
600 µl
We find decanting more effective than pipetting. If the pipette tip touches the phase lock gel, it makes a huge mess.
This should be performed inside the fume hood.
?. [Phenol chloroform extraction]
Centrifuge the phase lock tube at  for . Phase lock layer should now separate aqueous and organic layers.
Centrifuge: 16000 34
?. [Phenol chloroform extraction]
Mix by placing tubes on a rocker at medium speed for  .
?. [Phenol chloroform extraction]
Quickly decant the aqueous (top) layer into a fresh 2.0 mL LoBind tube.
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 chloform significantly 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 but 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.
This should be performed inside the fume hood.
?. [DNA precipitation, wash, and resuspension]
Chill 100% ethanol on ice and make  of fresh 70% ethanol using nuclease-free water.
1 mL
?. [DNA precipitation, wash, and resuspension]
Add 0.1 volume (typically ) of 3M sodium acetate to the extract from Step 24. Gently swirl to mix.
50 µl
?. [DNA precipitation, wash, and resuspension]
Add 2-2.5 volumes (typically ) of cold 100% ethanol to the tube, and mix with careful swirling and gentle rocking. DNA should slowly precipitate into a single white stringy clump, and un-precipitated DNA should be visible as shimmering strands at the bottom of the tube that are attached to the white clump.
1200 µl
If the extraction tube turns cloudy, it is likely salt precipitation because the solution is too nonpolar and not DNA. Add water dropwise with thorough mixing and the solution should clear up.
?. [DNA precipitation, wash, and resuspension]
Using a P200 pipette and a wide-bore tip, transfer the stringy clump to a fresh 1.5 mL LoBind tube.
This step can be somewhat tricky. The DNA clump can get stuck to your pipette tip and be very difficult to get off. Making sure that the DNA is fully precipitated (no un-precipitated strands present) and quick pipetting helps prevent this. PosResuspending DNAition the pipette tip right above the clump and aspirate quickly to bring the clump into the tip. Using a similar quick motion, dispense the DNA clump and liquid into the fresh 1.5 mL tube.
?. [DNA precipitation, wash, and resuspension]
Centrifuge the tube at  for  to pellet the DNA.
Centrifuge: 2000 34
?. [DNA precipitation, wash, and resuspension]
While being careful not to disturb the pellet, pipette off the ethanol.
?. [DNA precipitation, wash, and resuspension]
Add  of 70% ethanol to wash the DNA. Gently swirl to mix.
200 µl
?. [DNA precipitation, wash, and resuspension]
Centrifuge the tube at  for .
Centrifuge: 2000 34
?. [DNA precipitation, wash, and resuspension]
While being careful not to disturb the pellet, pipette off the ethanol.
I usually spin the tube down briefly (~2 sec) and use a P10/P20 to grab the last bit of ethanol in the tube.
?. [DNA precipitation, wash, and resuspension]
Wash the pellet once more:  .
?. [DNA precipitation, wash, and resuspension]
Allow the DNA to air dry right until the moment it becomes translucent (usually ). Do not over-dry the pellet.
?. [DNA precipitation, wash, and resuspension]
Resuspend in  1X TE buffer and incubate at  for .
65 µl
50 °C
?. [DNA precipitation, wash, and resuspension]
Briefly spin down tube to gather any condensation and store at .
4 °C
?. [DNA resuspension]
Keep the DNA at 4C for at least 1 week to obtain proper resuspension. Every ~48 hours, mix the DNA gently with a P1000 or P200 pipette with a normal tip. This will encourage DNA to resuspend and make sure it is adequately sheared for library prep. We usually mix three times:1. 10X with a P10002. 5X with a P10003. 5X with a P200
While it is possible to over-shear the DNA, under-shearing makes for much worse Nanopore runs because it makes the sample too viscous. More shearing may be needed depending on how much the sample was mixed during incubation, or how fresh the flies were.
?. [DNA resuspension]
Check sample concentration and quality of  aliquots using Qubit and Nanodrop.
1 µl
Ideally, this should Qubit at >75 ng/uL and have Nanodrop ratios of 260/280 >1.8 and 260/230 >2.0.
?. [DNA repair and end-prep]
Thaw NEBNext repair and dA-tailing mixes and buffers from the Nanopore Companion Module. Mix buffers by vortexing and mixes by flicking. Spin down tubes and keep chilled on ice.
?. [DNA repair and end-prep]
Add 3000-4000 ng of HMW DNA (typically ) to a PCR tube. Dilute the HMW DNA with water to a final volume of . Add  of FFPE DNA repair buffer,  of end-prep reaction buffer,  of FFPE DNA repair mix, and  of end-prep reaction mix to the tube. Mix tube with gentle flicking (or very gentle pipetting with a cut-off P200 tip).
30 µl
48 µl
3.5 µl
3.5 µl
2 µl
3 µl
?. [DNA repair and end-prep]
In a thermal cycler, incubate at  for  then  for . After this, sample can be held at  temporarily until ready to proceed.
20 °C
65 °C
4 °C
?. [DNA repair and end-prep]
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  SRE buffer. Using a wide-bore P200 tip, quickly but gently mix the tube. The precipitation buffer described here can be used in place of the SRE buffer but is not as effect at removing small DNA fragments as SRE.
60 µl
?. [DNA repair and end-prep]
Centrifuge the sample at  for  or until DNA has pelleted and solution is no longer viscous. Meanwhile, prepare  fresh 70% ethanol with nuclease-free water, or  of PB diluted 1:1 with nuclease-free water.
Centrifuge: 10000 34
500 µl
500 µl
?. [DNA repair and end-prep]
Pipette off the supernatant, taking care not to disturb the DNA pellet.
?. [DNA repair and end-prep]
Add  of 70% ethanol or diluted PB solution. Pipette slowly, with the tip touching the front wall of the tube, so that the pellet is not disturbed.
150 µl
?. [DNA repair and end-prep]
Centrifuge at  for .
Centrifuge: 10000 34
?. [DNA repair and end-prep]
Pipette off the supernatant, taking care not to disturb the DNA pellet. Make sure all the supernatant is removed and only the pellet remains.
?. [DNA repair and end-prep]
Repeat steps 46-48 once more.
?. [DNA repair and end-prep]
Resuspend pellet in  EB.
31 µl
?. [DNA repair and end-prep]
Incubate the tube on the heat block at  for . Briefly spin down the tube to collect condensation. Incubate at least  and preferably for twice that time at .
50 °C
4 °C
?. [Adapter ligation]
Thaw AMX, T4 ligase, LNB, and LFB from the NEBNext Nanopore Companion Module and the Nanopore LSK109 kit. Mix AMX, T4 ligase, and LFB by flicking. Mix LNB by pipetting. Briefly spin the tubes down and keep chilled on ice.
?. [Adapter ligation]
Add  prepared DNA sample (the extra  can be used to Qubit),  AMX, and  T4 ligase to a fresh 1.5 mL DNA LoBind tube. Gently flick the tube to mix.
30 µl
1 µl
2.5 µl
5 µl
?. [Adapter ligation]
Add  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.
12.5 µl
?. [Adapter ligation]
Incubate the reaction mixture at  for .
0 Room temperature
?. [Adapter ligation]
Centrifuge the tube at  for  to pellet the DNA.
Centrifuge: 10000 34
?. [Adapter ligation]
Pipette off the supernatant, being careful not to disturb the DNA pellet.
?. [Adapter ligation]
Add  of LFB to the tube. SFB or a 1:1 dilution of PB can be used here (similar to step 46).
100 µl
DO NOT USE ETHANOL TO WASH PREPARED LIBRARY. It will denature the motor protein.
?. [Adapter ligation]
Centrifuge the sample at  for .
Centrifuge: 10000 34
?. [Adapter ligation]
Being careful not to disturb the pellet, pipette off all the supernatant.
?. [Adapter ligation]
Repeat steps 58-60 to wash the pellet once more.
?. [Adapter ligation]
Resuspend pellet in  EB.
31 µl
?. [Adapter ligation]
Incubate library on the heat block at  for . Briefly spin down the tube to collect condensation then incubate for at least  before the next step.
40 °C
?. [(Optional) Library size selection with SRE buffer]
Quantify library concentration using  of the prepared library with Qubit. This step should not be performed unless library concentration is greater than . If the concentration is greater than  the library should be diluted to improve size selection performance.
1 µl
?. [(Optional) Library size selection with SRE buffer]
Add an equal volume () of SRE buffer to the library and gently pipette mix using a wide-bore tip.
30 µl
?. [(Optional) Library size selection with SRE buffer]
Add  of prepared library to a fresh 1.5 mL DNA LoBind tube.
30 µl
?. [(Optional) Library size selection with SRE buffer]
Centrifuge at .
Centrifuge: 10000 34, 30 min
?. [(Optional) Library size selection with SRE buffer]
Pipette off the supernatant, being careful not to disturb the DNA pellet at the bottom of the tube.
?. [(Optional) Library size selection with SRE buffer]
Add 100 uL of LFB, SFB, or 1:1 diluted PB (similar to step 46) to wash the pellet. It does not really matter which one is used.
DO NOT USE ETHANOL TO WASH PREPARED LIBRARY. It will denature the motor protein.
?. [(Optional) Library size selection with SRE buffer]
Centrifuge tube at  for .
Centrifuge: 10000 34
?. [(Optional) Library size selection with SRE buffer]
Being careful not to disturb the pellet, pipette off all the supernatant.
?. [(Optional) Library size selection with SRE buffer]
Repeat steps 69-71 to wash the pellet once more.
?. [(Optional) Library size selection with SRE buffer]
Resuspend the pellet in  EB.
31 µl
?. [(Optional) Library size selection with SRE buffer]
Incubate the tube on the heat block at   for . Briefly spin down the tube to collect condensation, and incubate at least  at  before sequencing.
40 °C
4 °C
?. [Tips for sequencing the library]
Quantify the concentration of  library with Qubit. We usually end up with  -  of total library at this stage.
1 µl
1000 ng
2000 ng
?. [Tips for sequencing the library]
With a cut off P200 tip, transfer about  of prepared library to a fresh 1.5mL LoBind tube. This should not exceed  in volume.
350 ng
35 µl
To maximize throughput and read length, it is critical to load enough library that flow cell pores will be occupied but not so much that they are oversaturated. The molar concentration of the library is a function of the fragment lengths so it is difficult to say exactly how much library to load. The average library prepared in this manner usually sequences well when   to   of DNA is loaded. Note that flow cells need to be flushed and reloaded so we usually aim to have at least 3 library loads.
?. [Tips for sequencing the library]
Add an equal volume of SQB from the LSK109 kit to the tube. Then, add FB from the marked tube (the one that we are not going to prepare the priming mix with) to a final volume of .For example, if  of  library was transferred in step 77, add  of SQB and  FB to the tube.
70 µl
10 µl
10 µl
50 µl
?. [Tips for sequencing the library]
Thaw 1 tube SQB (SQK-LSK109), 2 tubes FB (EXP-FLP002), and 1 tube FLT (EXP-FLP002). Mix SQB and FB by flicking. Mix FLT with a pipette. Keep reagents on ice until ready to sequence.
We recommend marking one tube of FB to use as dilution buffer for subsequent runs. Only one tube should be used to prepare the priming mix.
The FB must be from the EXP-FLP002 kit. This will not work with version 1 of the kit.
?. [Tips for sequencing the library]
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.
?. [Tips for sequencing the library]
Over the course of a sequencing run, pores will get clogged and become inactive. It is essential to flush the flow cell at 10-14 hour intervals to make these pores available again. We recommend Nanopore's Flow Cell Wash Kit (EXP-WSH003).