Button mushroom (Agaricus bisporus) DNA
Oxford Nanopore
Button mushroom (Agaricus bisporus) DNA
FOR RESEARCH USE ONLY
Contents
Introduction
Materials
Method
Results
Sequencing performance
Introduction
This protocol describes a method to extract high molecular weight genomic DNA from button mushrooms (Agaricus bisporus) using nuclei isolation, performed as described in Zhang, M. et al., 2012, followed by QIAGEN Genomic-tip purification. Prior to sequencing, 8 µg of genomic DNA was size-selected using the size selection of HMW DNA by semi-selective DNA precipitation protocol. Sequencing performance was determined using the MinION, using the Ligation Sequencing Kit to generate sequencing libraries.
Materials
For nuclei isolation:
- 8 g mushroom gills
- TissueRuptor II and disposable probes
- Trizma base
- KCl
- Na2EDTA
- Spermidine trihydrochloride
- Spermine tetrahydrochloride
- NaOH
- B-mercaptoehanol
- Triton X-100
- Sucrose
- ddH20
- Miracloth
- Cheesecloth
- Funnel
- 50 ml Falcon tubes
- Refrigerated centrifuge with capacity for 50 ml Falcon tubes
- Ice bucket with ice
- P20, P100, P200 and P1000 pipettes, tips and wide-bore pipette tips
For gDNA extraction and purification:
- QIAGEN Blood and Cell Culture DNA Midi Kit
- Isopropanol
- 70% ethanol
- Proteinase K
- Vortex
- 50 ml Falcon tubes
- Refrigerated centrifuge with capacity for 50 ml tubes
- Incubator or water bath with agitation capability and temperature control for 50°C
- TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
- P20, P100, P200 and P1000 pipettes, tips and wide-bore pipette tips
Method
Nuclei isolation:
- Prepare 10X Homogenisation Buffer (HB) stock as below. Adjust the pH to 9.0-9.4 with NaOH, and store the solution at 4°C. Tip: When developing this protocol, we made up 1 litre of HB stock and stored it at 4°C for up to a year.
HB stock:
| Reagents | Concentration (mM) |
|---|---|
| Trizma base | 100 mM |
| KCl | 800 mM |
| Na2EDTA | 100 mM |
| Spermidine trihydrochloride | 10 mM |
| Spermine tetrahydrochloride | 10 mM |
- Prepare approximately 90 ml of 1x Homogenisation Buffer (HB) working solution. Keep the solution at 4°C.
HB working solution:
| Reagents | Concentration |
|---|---|
| HB stock | 1x |
| Sucrose | 0.5 M |
Prepare two 50 ml tubes per sample and add 20 ml of HB working solution to each.
Add 4 g of mushroom gills to each tube.
Homogenise the mushroom sample using the TissueRuptor II at the lowest speed. This will take between 45 seconds to a minute to homogenise
Add another 20 ml of HB working solution to each tube and invert a few times.
Transfer the tubes to a fume hood and continue the next steps inside the hood until step 20 due to the β-mercaptoethanol toxicity.
Add 0.15% (v/v) of β-mercaptoethanol and invert the tubes 10 times to mix.
Incubate the tubes in a HulaMixer (or equivalent) at 4°C for 10 minutes. If there is no mixer available at 4°C, keep the tubes on ice and gently invert them five times every minute.
Put a funnel on top of two fresh 50 ml Falcon tubes and add a layer of miracloth and two layers of cheesecloth.
Pass the solutions from step 9 through the funnel prepared in step 10. We recommend squeezing the miracloth to maximise the nuclei recovery.
Centrifuge the tubes at 4,000 x g for 20 minutes at 4°C.
Discard the supernatant.
Prepare approximately 45 ml of HB washing solution as below and cool to 4°C. HB washing solution:
| Reagents | Concentration |
|---|---|
| HB working solution | 1X |
| β-mercaptoethanol | 0.15% (v/v) |
| Triton X-100 | 0.2% (v/v) |
Add 1 ml HB washing solution to the pellets and gently resuspend the pellets by pipetting up and down with a wide-bore pipette tip.
Add 9 ml HB washing solution to both tubes, and gently invert 10 times to mix.
Centrifuge the tubes at 3,100 x g for 15 minutes at 4°C.
Discard the supernatant.
Repeat steps 15-18.
Add 500 µl of 1X HB working solution and gently resuspend the pellet with a wide-bore pipette tip.
DNA extraction:
To each tube with isolated nuclei, add 10 ml of buffer G2 and 100 µl of proteinase K,before inverting the tube 10 times to mix.
Incubate at 50°C for 1 hour with gentle mixing (100 RPM). The tube contents should appear homogeneous but if solid particles are still visible, centrifuge the tubes for a minute at 2,000 x g and transfer the supernatant to fresh tubes.
Equilibrate a Genomic-tip 100/G with 4 ml of buffer QBT.
Pour one of the tubes with lysate into the Genomic-tip 100/G.
Once the content of the first tube has passed through, pour the second tube into the Genomic-tip 100/G.
Purify the lysate according to the standard QIAGEN protocol (steps 3-5, pages 50-51).
After adding the isopropanol, invert the tubes 10 times and incubate it overnight at −20°C.
Centrifuge the tube at 4,000 x g for 30 minutes at 4°C.
Discard the supernatant and add 5 ml of ice-cold 70% ethanol.
Invert the tube five times and centrifuge at 4,000 x g for 3 minutes at 4°C.
Discard the supernatant and use a clean tissue to dry the walls of the tube.
Elute the pellet in 150 µl TE buffer.
Optional step: Take 60 µl of the gDNA and size select your sample using the size selection protocol. About 60% of gDNA is expected to be recovered.
Results
- Yield: 15-25 µg
- A260/280: 1.92
- A260/230: 2.14
Sequencing performance
The library for nanopore sequencing was prepared using the Ligation Sequencing Kit.
The flow cell was washed using the Flow Cell Wash Kit (EXP-WSH004) and the library re-loaded after ~20 hours of sequencing to maximise flow cell output. Read length profile:
