Mar 22, 2022

Public workspaceIsolation of high molecular weight genomic DNA from Entomophthora muscae

DOI
dx.doi.org/10.17504/protocols.io.rm7vzyje2lx1/v1
  • 1Harvard University;
  • 2New York Genome Center
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzyje2lx1/v1
Protocol Citation: Carolyn Elya, Emily Lee 2022. Isolation of high molecular weight genomic DNA from Entomophthora muscae. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzyje2lx1/v1
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: March 22, 2022
Last Modified: March 22, 2022
Protocol Integer ID: 59750
Funders Acknowledgement:
HHMI
Grant ID: Hanna Gray Fellowship
Abstract
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.
Protocol materials
ReagentGraces Insect MediumThermo FisherCatalog #11605094
Step 1
ReagentRNase AFischer ScientificCatalog #FEREN0531
Step 14
Reagentλ DNA-Mono Cut MixNEBCatalog #N3019S
Step 36.1
ReagentSYBR™ Safe DNA Gel StainThermo FisherCatalog #S33102
Step 36.1
Reagent50 ml conical tubes
Step 3
ReagentGlycogenFisher ScientificCatalog #FERR0551
Step 27
ReagentGenomic DNA ReagentsAgilent TechnologiesCatalog #5067-5366
Step 36.2
ReagentAxygen MaxyClear TubesVWR InternationalCatalog #22234-046
Step 5
ReagentKontes Pellet Pestle 1.5 mLVWR InternationalCatalog #KT-749521-1590
Step 11
ReagentGenomic DNA ScreenTapeAgilent TechnologiesCatalog #5067-5365
Step 36.2
ReagentFetal Bovine SerumThermo FisherCatalog #10437010
Step 1
ReagentFalcon 25cm^2 flaskCorningCatalog #353014
Step 1.1
ReagentProteinase KNEBCatalog #P8107S
Step 12
ReagentQubit™ dsDNA HS Assay kitThermo FisherCatalog #Q32851
Step 35
Fungal culture
Fungal culture
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 ReagentGraces Insect MediumThermo FisherCatalog #11605094 with added 5% FBS (e.g., ReagentFetal Bovine SerumThermo FisherCatalog #10437010 ) , which will henceforth be referred to as Grace's + 5% FBS.


CITATION
Hajek AE, Papierok B, Eilenberg J.. Chapter IX - Methods for study of the Entomophthorales. Lacey LA, editor. Manual of Techniques in Invertebrate Pathology (Second Edition).
LINK
doi:10.1016/B978-0-12-386899-2.00009-9


From -80C glycerol stock
Transfer Amount20 mL of Grace's + 5% FBS to ReagentFalcon 25cm^2 flaskCorningCatalog #353014 and allow to come to room temperature.
Retrieve one Amount1.5 mL -Amount2 mL aliquot from Temperature-80 °C and rapidly thaw entire aliquot at Temperature37 °C .
Transfer entire contents to prepare tissue culture flask.
15m
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 Duration02:00:00 and no more than Duration15:00:00 ) 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 Duration48:00:00 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 Amount20 mL .

2d 17h
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.
1w
Harvest cells
Harvest cells
Transfer at least Amount20 mL of in vitro culture to sterile Reagent50 ml conical tubesContributed by users and spin 3500 rpm, Duration00:15:00 , 4C. This will produce a somewhat loose pellet.

15m
Decant most of supernatant, leaving loose pellet behind.
Resuspend the cells in any remaining supernatant (gently flick tube or pipette up and down with micropipettor) and transfer cell sludge to ReagentAxygen MaxyClear TubesVWR InternationalCatalog #22234-046

30s
Spin eppendorf tube forDuration00:05:00 at maximum rpm at TemperatureRoom temperature . Tight pellet will result.

5m
Aspirate supernatant and discard.
30s
Flash freeze tube contents using liquid nitrogen or dry ice + ethanol bath.
5m
If you are not ready to proceed with DNA extraction, cells should be stored at Temperature-80 °C .

DNA extraction - Day 1
DNA extraction - Day 1
This DNA extraction is based on methods reported in Bulat et al, 1998
CITATION
Bulat SA, Lübeck M, Mironenko N, Jensen DF, Lübeck PS. UP-PCR analysis and ITS1 ribotyping of strains of Trichoderma and Gliocladium.. Mycol Res.
LINK
10.1017/S0953756297005686


5m
Remove samples from cold bath or Temperature-80 °C and thaw at TemperatureRoom temperature .
In a fume hood, homogenize each sample with a ReagentKontes Pellet Pestle 1.5 mLVWR InternationalCatalog #KT-749521-1590 then rinse pestle with Amount400 µL Buffer A.

5m
AddAmount13 µL of proteinase K (ReagentProteinase KNEBCatalog #P8107S 20 mg/mL) to each sample to reach 600 ug/mL.

2m
This assumes pellet volume was approximately Amount20 µL . If substantially deviated from this, adjust volume of proteinase K as needed to achieve desired concentration of 600 ug/mL.

Gently pipette suspension and mix further by gentle inversion, then briefly spin to collect all liquid in bottom of tube.
1m
Add 4.3 uL RNase A ReagentRNase AFischer ScientificCatalog #FEREN0531 to each sample, mixing thoroughly by pipetting or gentle inversion, then briefly spin to collect all liquid in bottom of tube.

2m
Incubate Duration00:30:00 atTemperature37 °C .

30m
Store at Temperature4 °C DurationOvernight .

30m
DNA extraction - Day 2
DNA extraction - Day 2
Retrieve samples from fridge and put in chemical safety hood. Allow to come to TemperatureRoom temperature before continuing.

18h
Adjust salt concentration to 1 M NaCl of each sample by adding Amount104 µL 4.4M NaCl, mixing gently by inversion.

1m
Add Amount500 µL of 24:1 chloroform:octanol to each sample and invert to mix.

2m
Incubate Duration00:15:00 at TemperatureRoom temperature .

15m
Spin Duration00:02:00 , 12,000xg, TemperatureRoom temperature

2m
Remove aqueous layer (i.e. top layer) and transfer to new tube.
5m
If volume in new tube less than Amount500 µL , add 1 M NaCl to bring up to Amount500 µL .

1m
Sticky aqueous layer is a good sign!
Repeat steps 18) to 21) as necessary until you arrive with a non-cloudy aqueous layer. This may need to be repeated several times.
Spin out any residual protein via spin for Duration00:02:00 , 12,000xg, TemperatureRoom temperature .

2m
Using a WIDE BORE TIP (cut P200 tip at first line with clean razor blade to generate wide opening) , transfer supernatant to new tube.
5m
Determine volume of supernatant using gradations on side of tube in combination with pipetting with wide-bore tip, as needed.
1m
Add Amount3 µL of glycogen (ReagentGlycogenFisher ScientificCatalog #FERR0551 ) to solution and mix gently by rocking back and forth.

2m
AddAmount0.6 vol of isopropanol (IPA) to supernatant. Invert GENTLY to mix and incubate at RT for Duration00:30:00 .

30m
Spin Duration00:10:00 ,max rpm, TemperatureRoom temperature to pellet out DNA.

10m
Wash pellet with Amount1 mL of 70% ethanol.

1m
Spin Duration00:02:00 , max rpm, RT to re-pellet.

2m
Remove all supernatant with P1000/P200 and air-dry ~Duration00:10:00 on kimwipe.

10m
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 (Amount50 µL -Amount100 µL ).

2m
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
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
35m
35m
Check polysaccharide and RNA contamination
Equipment
NanoDrop™ One UV-Vis Spectrophotometer
NAME
spectrophotometer
TYPE
Thermo Scientific
BRAND
ND-ONE-W
SKU
https://www.thermofisher.com/order/catalog/product/ND-ONE-W
LINK
Sample Volume (Metric): Minimum 1µL; Spectral Bandwidth: ≤1.8 nm (FWHM at Hg 254 nm); System Requirements: Windows™ 8.1 and 10, 64 bit; Voltage: 12 V (DC); Wavelength Range: 190–850 nm
SPECIFICATIONS

  1. Apply Amount1.5 µL -Amount2 µL of gDNA preparation to nanodrop and read A260/280 (RNA contamination); A260/230 (polysaccharide contamination).

5m
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.
Check DNA concentration using ReagentQubit™ dsDNA HS Assay kitThermo FisherCatalog #Q32851
Equipment
new equipment
NAME
Qubit 2.0 Fluorometer instrument
BRAND
Q33226
SKU
with Qubit RNA HS Assays
SPECIFICATIONS

30m
Prepare triplicate 1/10 and 1/100 dilution samples for Qubit per manufacturer protocol.
Check size distribution

This can be done via agarose gel or
Equipment
4200 TapeStation System
NAME
Electrophoresis tool for DNA and RNA sample quality control.
TYPE
TapeStation Instruments
BRAND
G2991AA
SKU
https://www.agilent.com/en/product/automated-electrophoresis/tapestation-systems/tapestation-instruments/4200-tapestation-system-228263
LINK

Gel method:
  1. Prepare 0.4% agarose gel in 1x TAE with NO intercalating agent.
  2. Use the highest grade agarose you have access to!
  3. Add back water after microwaving to maintain correct percentage of agarose.
  4. Run out sample with high molecular weight fragment ladder (e.g.Reagentλ DNA-Mono Cut MixNEBCatalog #N3019S ) 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).
  5. Stain with ReagentSYBR™ Safe DNA Gel StainThermo FisherCatalog #S33102 in water (1x solution) for 15 minutes at RT with gentle shaking.
  6. Destain with water for 15 minutes at RT with gentle shaking.
  7. Image with gel doc.


Tape Station method:
  1. Bring ReagentGenomic DNA ReagentsAgilent TechnologiesCatalog #5067-5366 & ReagentGenomic DNA ScreenTapeAgilent TechnologiesCatalog #5067-5365 from 4C to RT for at least 30 min before starting.
  2. Prepare 10-100 ng/uL dilution of your DNA.
  3. Follow TapeStation Genomic DNA ScreenTape instructions to load samples and run.


You could also use the
Equipment
2100 Bioanalyzer Instrument
NAME
Sizing, quantification, and sample quality control of DNA, RNA, and proteins on a single platform
TYPE
Agilent Technologies
BRAND
G2939BA
SKU
for this analysis, but it's a pain in the butt compared to the TapeStation.
Citations
Step 1
Hajek AE, Papierok B, Eilenberg J.. Chapter IX - Methods for study of the Entomophthorales
doi:10.1016/B978-0-12-386899-2.00009-9
Step 9
Bulat SA, Lübeck M, Mironenko N, Jensen DF, Lübeck PS. UP-PCR analysis and ITS1 ribotyping of strains of Trichoderma and Gliocladium.
10.1017/S0953756297005686