TITLE: Isolation of supernumerary mini-chromosomes from fungi for enrichment sequencing
AUTHORS: Thorsten Langner, Adeline Harant, Sophien Kamoun

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Fungal genomes are highly dynamic and often contain supernumerary mini-chromosomes. However, our knowledge about the role of mini-chromosomes in dynamic genome rearrangements and evolution is scarce. Mini-chromosomes are usually smaller then core-chromosomes and are variable in size and numbers between individuals of a species. Mini-chromosome occurence in fungi is well documented since decades, but many genomic projects have neglected them, mainly because it was technically challenging to identify them in whole genome assemblies. </div><div class = "text-block">Here we present a protocol for isolation of mini-chromosomes for enrichment sequencing. This allows identification of mini-chromosomes in whole genome assemblies and thus comparative genomics analyses of core- and mini-chromosomes.</div><div class = "text-block"><div class = "justify" style = "text-align:justify"></div><style>
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[STEPS]
?. [Preparation of the protoplasts]
Prepare a fresh culture of M. oryzae on complete medium agar plates.
?. [Preparation of the protoplasts]
Incubate at 24°C for 7 days.
?. [Preparation of the protoplasts]
Inoculate 150 ml of YG medium in a 500 ml Erlenmeyer flask with 8 pieces (~0.5 x 0.5 cm) of 7 days old M. oryzae mycelium.
?. [Preparation of the protoplasts]
Shake at 120 rpm at 24°C for 3 days.
?. [Preparation of the protoplasts]
Prepare lysing enzymes solution and filter sterilise it.
?. [Preparation of the protoplasts]
Filter culture through 2 layers of miracloth and transfer mycelia to a 50 ml falcon tube or 100 ml beaker.
Put the miracloth in a funnel on top of a bottle to collect the media. Remove as much media residues as possible by squeezing gently.
?. [Preparation of the protoplasts]
Add 15mL of lysing enzyme solution to the mycelia.
?. [Preparation of the protoplasts]
Place the tube on a horizontal shaker at room temperature for 2 to 4 h. Shake at 30 rpm.
Check the progress of protoplast formation under the microscope.
?. [Preparation of the protoplasts]
Filter protoplasts through 2 layers of miracloth and collect the flow through.
Place the miracloth in a funnel on top of a 50mL falcon.
?. [Preparation of the protoplasts]
Centrifuge flow through at 1500 rpm at room temperature for 10 min. Discard the supernatant.
Be careful not to dislodge the pellet. Use a pipette to remove supernatant.
?. [Preparation of the protoplasts]
Add 25 mL of Sorbitol 1 M and centrifuge again. Discard the Supernatant and repeat (2 wash steps in total).
?. [Preparation of the protoplasts]
After the washing steps, remove as much of the supernatant as possible using a pipette. Do not disrupt the pellet.
?. [Preparation of the protoplasts]
Add 100-200 µl of 1M Sorbitol/50mM EDTA and resuspend the protoplast with a cut 1mL tip.
The protoplasts are very fragile! Pipette very slowly up and down or shake very gently to not damage the protoplasts.
?. [Preparation of the protoplasts]
Check quality and concentration of protoplast under the microscope using a hemocytometer.
To visualize chromosome size fragments after CHEF gel electrophoresis and for subsequent DNA extraction, high protoplast concentration is required. Concentration of protoplasts should be ~ 3 x 109 protoplasts/ml.
?. [Preparation of agarose gel plugs for contour-clamped homogenous electric field (CHEF) gel electrophoresis]
Mix 1/3 volume of protoplasts with 2/3 volume of 1% megabase agarose in 1M Sorbitol/50mM EDTA at 45°C.
Prepare the gel in advance and let it cool down in a water bath at 55°C.
?. [Preparation of agarose gel plugs for contour-clamped homogenous electric field (CHEF) gel electrophoresis]
Immediately after mixing, pipette protoplast mix into Bio-Rad plug mould (#1703713) and let solidify on ice for at least 30 min.
Make sure to proceed quickly before the gel can solidify.Tip: Pipette the liquid gel while keeping the tube in the water bath. Mix briefly by pipetting up and down twice and directly transfer the solution in the plug mould. Alternatively, prepare protoplast/agarose gel mix in 1.5 ml Eppendorf tube and cut to size after the mix has solidified. For DNA extraction more than one plug might be required.
?. [Preparation of agarose gel plugs for contour-clamped homogenous electric field (CHEF) gel electrophoresis]
Remove the protoplast-agarose plug from the mould. Transfer to a 1.5 ml Eppendorf tube and add 1 ml NDS buffer containing 200 µg/ml proteinase K.
?. [Preparation of agarose gel plugs for contour-clamped homogenous electric field (CHEF) gel electrophoresis]
Incubate at 50°C for 48 h.
?. [Preparation of agarose gel plugs for contour-clamped homogenous electric field (CHEF) gel electrophoresis]
Remove the NDS buffer, add 1 ml of fresh 50mM EDTA and shake slowly at room temperature for 30 min to 1 h. Repeat 3 times.
Washing steps can be carried out on a thermomixer set to 22°C and 200 rpm.
?. [Preparation of agarose gel plugs for contour-clamped homogenous electric field (CHEF) gel electrophoresis]
Carefully remove EDTA solution and store the plugs in NDS buffer (without proteinase K) at 4°C until separation by CHEF gel electrophoresis.
?. [CHEF gel electrophoretic separation of mini-chromosomes]
Prepare 1% megabase agarose gel in 0.5% buffer (TAE or TBE buffer) and let solidify.
Use TBE buffer for better resolution during long runs and TAE buffer for extraction of DNA for library preparation. The borate in the TBE buffer can inhibit downstream enzymatic reactions.
?. [CHEF gel electrophoretic separation of mini-chromosomes]
Place the agarose plugs in the wells. Place the size markers.
Saccharomyces cerevisiae and Schizosaccharomyces pombe chromosomes are commercially available and can be used as size markers (Biorad, #1703633, #1703605).
?. [CHEF gel electrophoretic separation of mini-chromosomes]
a.Initial switch interval      120s        b.End switch interval         3600s        c.Voltage                               1.8 - 2V/cm        d.Run time                             96 h        e.Temperature                     14°C
Prepare the system early and let the circulating buffer cool down to 14°C for at least 30 min. Do not move the system during the run, constant buffer flow and temperature are important for good resolution. We developed this protocol on a Biorad CHEF DRII system. The settings specified above are optimized to separate chromosomes between 400 kb and 3 MB.
?. [CHEF gel electrophoretic separation of mini-chromosomes]
To visualize chromosome sized fragments, place gel into ethidium bromide solution (1 µg/ml in 0.5% buffer) for 30 min and wash in 0.5% buffer (TAE or TBE) for 10 min and observe on a UV transilluminator.
?. [Extraction of chromosome sized fragments from agarose gel by electroelution]
Cut bands out of the gel using a scalpel and store gel slices in Eppendorf tubes at 4°C.
Remove as much of the gel as possible. The gel slice should have clean edges to prevent contamination of the buffer with agarose in the next step.
?. [Extraction of chromosome sized fragments from agarose gel by electroelution]
Moisturize a D-Tube Dialyzer Midi 3.5 kDa MWCO (Merck, #71506) with 800 µl ddH2O for 1 min. Check membrane for leaks.
?. [Extraction of chromosome sized fragments from agarose gel by electroelution]
Remove water with a pipette and place agarose gel slice into the chamber.
Do not touch the membrane with the pipette tip.
?. [Extraction of chromosome sized fragments from agarose gel by electroelution]
Fill the chamber of the D-Tube with 0.5% TAE buffer and close the lid tightly. Place tube in electrophoresis rack (D-Tube Electroelution Accessory Kit, Merck, #71511). Place in electrophoresis chamber.
?. [Extraction of chromosome sized fragments from agarose gel by electroelution]
Run electroelution in 0.5% TAE buffer at 90 V for 3 h at room temperature.
Membranes should face in running direction. The chromosomal DNA will migrate out of the gel and gets collected in the buffer. Some DNA might stick to the membrane.
?. [Extraction of chromosome sized fragments from agarose gel by electroelution]
Carefully resuspend DNA by pipetting up and down close to the membrane.
Do not touch the membrane with the pipette tip to prevent sample loss. Do not touch the gel slice with the pipette tip as this can lead to agarose contamination which can decrease efficiency during downstream enzymatic reactions or library preparation.
?. [Extraction of chromosome sized fragments from agarose gel by electroelution]
Proceed with Library preparation according to manufacturer’s instructions. We tested this protocol using a Nextera low input library preparation kit (Illumina) with custom barcoding primers and sequenced mini-chromosome derived libraries on a Nextseq 500 system.
?. [Extraction of chromosome sized fragments from agarose gel by electroelution]
Transfer DNA solution to a 1.5 ml eppendorf tube and concentrate in a speedvac concentrator.
Concentrate DNA solution to a volume of 20-50 µl. For library preparation adjust concentration following manufacturer's instructions. DNA concentration of 0.5 ng/µl is sufficient for low input library preparations. We recommend to use fluorometric methods to measure DNA concentration.