TITLE: Isolation of high molecular weight genomic DNA from Entomophthora muscae
AUTHORS: Carolyn  Elya

[DESCRIPTION]
Obtaining quality genomic material from Entomophthoralean fungi has proven extremely difficult. This protocol describes a method that has been successfully used to obtain high molecular weight genomic DNA from the obligate fungal pathogen Entomophthora muscae for long-read sequencing and genome assembly. The protocol details quality control and quantification steps recommended before proceeding to downstream applications, such as sequencing.

[STEPS]
SECTION: Fungal culture
1. Fungal culture can be established from glycerol freezer stock or from a sporulating cadaver following methods outlined in Hajek et al, 2012. Cultures are grown in supplemented   with added 5% FBS (e.g.,  ) , which will henceforth be referred to as Grace's + 5% FBS.


 


SECTION: Fungal culture
1.1. From -80C glycerol stock
Transfer 20 mL of Grace's + 5% FBS to   and allow to come to room temperature. 
Retrieve one 1.5 mL-2 mL aliquot from -80 °C and rapidly thaw entire aliquot at 37 °C.
Transfer entire contents to prepare tissue culture flask.
SECTION: Fungal culture
1.2. From sporulating cadaver
Collect conidia via ascending method per Hajek et al, 2012. Briefly, using aseptic technique: 
-Adhere cadaver via ventral abdomen to the top of an inverted sterile petri dish and allow to sporulate for several hours (at least 120 min and no more than 900 min) onto the base of the dish. 
-Replace the top of the dish, invert the dish and cover spores with room temperature Grace's + 5% FBS. 
-Incubate dish for at least 2880 min at room temperature, monitoring for contamination. If the dish becomes uniformly cloudy at any point, it has been contaminated by something other than E. muscae.
-Once clumpy, protoplastic E. muscae growth can be observed, transfer culture to 25 cm^2 flask along with room temperature Grace's + 5% FBS to total final culture volume of 20 mL.

SECTION: Fungal culture
2. Incubate culture at room temperature until you reach late logarithmic growth. Culture morphology should still resemble heterogeneous small white clumps and lack obvious mycelial threads/tangles.
SECTION: Harvest cells
3. Transfer at least 20 mL of in vitro culture to sterile   and spin 3500 rpm, 15 min, 4C. This will produce a somewhat loose pellet.

SECTION: Harvest cells
4. Decant most of supernatant, leaving loose pellet behind.
SECTION: Harvest cells
5. Resuspend the cells in any remaining supernatant (gently flick tube or pipette up and down with micropipettor) and transfer cell sludge to  

SECTION: Harvest cells
6. Spin eppendorf tube for5 min at maximum rpm at -80 Room temperature. Tight pellet will result.

SECTION: Harvest cells
7. Aspirate supernatant and discard.
SECTION: Harvest cells
8. Flash freeze tube contents using liquid nitrogen or dry ice + ethanol bath.
SECTION: Harvest cells
8.1. If you are not ready to proceed with DNA extraction, cells should be stored at -80 °C. 

SECTION: DNA extraction - Day 1
9. This DNA extraction is based on methods reported in Bulat et al, 1998
 


SECTION: DNA extraction - Day 1
11. In a fume hood, homogenize each sample with a   then rinse pestle with 400 µL Buffer A.

SECTION: DNA extraction - Day 1
12. Add13 µLof proteinase K (   20 mg/mL) to each sample to reach 600 ug/mL.

SECTION: DNA extraction - Day 1
12.1. This assumes pellet volume was approximately 20 µL. If substantially deviated from this, adjust volume of proteinase K as needed to achieve desired concentration of 600 ug/mL.

SECTION: DNA extraction - Day 1
13. Gently pipette suspension and mix further by gentle inversion, then briefly spin to collect all liquid in bottom of tube.
SECTION: DNA extraction - Day 1
14. Add 4.3 uL RNase A   to each sample, mixing thoroughly by pipetting or gentle inversion, then briefly spin to collect all liquid in bottom of tube.

SECTION: DNA extraction - Day 1
15. Incubate 30 min at37 °C.

SECTION: DNA extraction - Day 1
16. Store at 4 °C 30 min.

SECTION: DNA extraction - Day 2
17. Retrieve samples from fridge and put in chemical safety hood. Allow to come to 4 Room temperature before continuing.

SECTION: DNA extraction - Day 2
18. Adjust salt concentration to 1 M NaCl of each sample by adding 104 µL 4.4M NaCl, mixing gently by inversion.

SECTION: DNA extraction - Day 2
19. Add 500 µL of 24:1 chloroform:octanol to each sample and invert to mix. 

SECTION: DNA extraction - Day 2
20. Incubate 15 min at 4 Room temperature. 

SECTION: DNA extraction - Day 2
21. Spin 2 min, 12,000xg, 4 Room temperature

SECTION: DNA extraction - Day 2
22. Remove aqueous layer (i.e. top layer) and transfer to new tube. 
SECTION: DNA extraction - Day 2
22.1. If volume in new tube less than 500 µL, add 1 M NaCl to bring up to 500 µL. 

SECTION: DNA extraction - Day 2
23. Repeat steps 18) to 21) as necessary until you arrive with a non-cloudy aqueous layer. This may need to be repeated several times.
SECTION: DNA extraction - Day 2
24. Spin out any residual protein via spin for 2 min, 12,000xg, 4 Room temperature. 

SECTION: DNA extraction - Day 2
25. Using a WIDE BORE TIP  (cut P200 tip at first line with clean razor blade to generate wide opening) , transfer supernatant to new tube.
SECTION: DNA extraction - Day 2
26. Determine volume of supernatant using gradations on side of tube in combination with pipetting with wide-bore tip, as needed.
SECTION: DNA extraction - Day 2
27. Add 3 µL of glycogen ( ) to solution and mix gently by rocking back and forth. 

SECTION: DNA extraction - Day 2
28. Add0.6 vol of isopropanol (IPA) to supernatant. Invert GENTLY to mix and incubate at RT for 30 min.

SECTION: DNA extraction - Day 2
29. Spin 10 min ,max rpm, 4 Room temperature to pellet out DNA.

SECTION: DNA extraction - Day 2
30. Wash pellet with 1 mLof 70% ethanol.

SECTION: DNA extraction - Day 2
22.2. Sticky aqueous layer is a good sign!
SECTION: DNA extraction - Day 2
31. Spin 2 min, max rpm, RT to re-pellet.

SECTION: DNA extraction - Day 2
32. Remove all supernatant with P1000/P200 and air-dry ~10 min on kimwipe.

SECTION: DNA extraction - Day 2
33. WITH WIDE BORE TIP (cut P200 tip at first line with clean razor blade to generate wide opening) resuspend each pellet in desired volume TE buffer (50 µL-100 µL).

SECTION: Quality control - Perform same day as finishing extraction to minimize freeze-thaw cycles. Performing in this order should give most salient information first and save time if prep looks bad
34. Check polysaccharide and RNA contamination
 

Apply 1.5 µL-2 µL of gDNA preparation to nanodrop and read A260/280 (RNA contamination); A260/230 (polysaccharide contamination).

SECTION: Quality control - Perform same day as finishing extraction to minimize freeze-thaw cycles. Performing in this order should give most salient information first and save time if prep looks bad
35. Check DNA concentration using  
 

SECTION: Quality control - Perform same day as finishing extraction to minimize freeze-thaw cycles. Performing in this order should give most salient information first and save time if prep looks bad
34.1. We want to see:
OD260/OD280 ratio between 1.8 and 2.0 (low protein contamination).
OD260/OD230 ratio between 2.0 and 2.2 (low carbohydrate contamination).
Nanodrop will also give you ballpark for concentration to help select appropriate dilutions for Qubit in next step.
SECTION: Quality control - Perform same day as finishing extraction to minimize freeze-thaw cycles. Performing in this order should give most salient information first and save time if prep looks bad
36. Check size distribution

This can be done via agarose gel or 
 

SECTION: Quality control - Perform same day as finishing extraction to minimize freeze-thaw cycles. Performing in this order should give most salient information first and save time if prep looks bad
36.1. Gel method:
Prepare 0.4% agarose gel in 1x TAE with NO intercalating agent.
Use the highest grade agarose you have access to!
Add back water after microwaving to maintain correct percentage of agarose.
Run out sample with high molecular weight fragment ladder (e.g. )   in 1x TAE at 3V/cm at RT until dye reaches ¾ way through gel (expect to take several hours (~4-5). Keep DNA in fridge/on ice during this time (DO NOT FREEZE).
Stain with    in water (1x solution) for 15 minutes at RT with gentle shaking.
Destain with water for 15 minutes at RT with gentle shaking.
Image with gel doc.


SECTION: Quality control - Perform same day as finishing extraction to minimize freeze-thaw cycles. Performing in this order should give most salient information first and save time if prep looks bad
36.2. Tape Station method:
Bring   &   from 4C to RT for at least 30 min before starting.
Prepare 10-100 ng/uL dilution of your DNA.
Follow TapeStation Genomic DNA ScreenTape instructions to load samples and run.


SECTION: Quality control - Perform same day as finishing extraction to minimize freeze-thaw cycles. Performing in this order should give most salient information first and save time if prep looks bad
36.3. You could also use the
 
for this analysis, but it's a pain in the butt compared to the TapeStation.
SECTION: Quality control - Perform same day as finishing extraction to minimize freeze-thaw cycles. Performing in this order should give most salient information first and save time if prep looks bad
35.1. Prepare triplicate 1/10 and 1/100  dilution samples for Qubit per manufacturer protocol.
SECTION: DNA extraction - Day 1
10. Remove samples from cold bath or -80 °C and thaw at -80 Room temperature.