TITLE: Cyanobacterial Growth, Harvest, and Genomic DNA Prep
AUTHORS: Ryan D Ward, Truc  Mai, Nicole  Pietrasiak

[DESCRIPTION]
This protocol is a method for the growth of terrestrial and freshwater cyanobacteria in liquid Z8 medium and the subsequent harvesting and genomic DNA extraction. Life history traits of these microorganisms such as firm cell walls, exopolysaccharide secretions, and variability in growth rates present challenges to studying their genotype. Our approach establishes a generalizable protocol to grow diverse cyanobacteria under the same conditions and a robust DNA extraction technique that produces high-quality low to medium molecule size DNA for Illumina genome sequencing.

[STEPS]
SECTION: Biomass growing conditions
1. Transfer100 mLsterile liquid Z8 media into a .
 
Label the flask with the strain ID. 
Inoculate with cyanobacterial specimen. 
Set vented cap with a 0.22µm pore-size PTFE membrane to the "open" position to allow for gas exchange.
Secure flask into an orbital shaker at beneath a fluorescent light at 35-40 μmole·m−2·s−1 and allow to grow until confluent or senescent.
Growth period may vary from 2 - 8 weeks depending on growth rate of cyanobacteria species.


SECTION: Harvesting and preserving specimens in a biological safety hood
2. 
Gently pour cyanobacterial biomass of a particular species into a labelled sterile 50 mL conical tube (Falcon® Centrifuge Tubes, Polypropylene, Sterile, Corning VWR Cat Nr. 21008-936).
SECTION: Harvesting and preserving specimens in a biological safety hood
3. Centrifuge5000 x g, 5 min 
Decant liquid Z8 media. 
Add50 mLliquid Z8 media to tube, cap, and shake vigorously to dislodge potential bacterial contaminants from the cyanobacteria biomass.
SECTION: Harvesting and preserving specimens in a biological safety hood
4. Repeat Step 3 three times
SECTION: Harvesting and preserving specimens in a biological safety hood
5. Add a final50 mLliquid Z8 media to tube.
Lightly close cap.
Wrap with foil and place in refrigerator at4 10for 24 hours to halt photosynthesis and chromosomal replication.

SECTION: Harvesting and preserving specimens in a biological safety hood
6. Carefully decant remaining liquid Z8 media without centrifugation.
Retrieve sample from tubes with a sterile inoculation loop (Globe Scientific Sterile Rigid Inoculating Loops ThermoFisher Cat Nr. 22-170-204) into sterile 1.5 mL Eppendorf tubes (Fisherbrand™ Locking-Lid Microcentrifuge Tubes with Polypropylene Snap-Cap Cat Nr.: 02-681-284) 
Centrifuge5000 x g, 3 min
Remove supernatant Z8 media with a P1000 pipette and discard.
SECTION: Harvesting and preserving specimens in a biological safety hood
7. Place tubes into liquid nitrogen for 5 minutes.
Immediately transfer to-80 °Cfreezer.
SECTION: Genomic DNA Prep
8. Thaw biomass on ice.
Transfer ca. 500 µLbiomass per PowerLyzer® bead beating tube using a sterile inoculation loop. 
Place bead tube and balance in the homogenizer.
 
Process samples during four stages, then allow the homogenizer to cool down before turning off. 
Apply the following setup:

5500 rpm, 45 s, pause5 s 
5500 rpm, 45 s, cool down5 min 
5500 rpm, 45 s, pause5 s 
5500 rpm, 45 s, cool down5 min 

SECTION: Genomic DNA Prep
9. 
Process samples with the  , following the manufacturer protocol, with a modified elution step as follows.


Place spin column into sterile 1.5 mL Eppendorf tube.


Transfer15 µLPowerLyzer® elution buffer to the center of the column membrane.
Incubate at room temperature for 3 minutes. 
Centrifuge1000 x g, 3 min 

Add an additional25 µL elution buffer to spin column.
Incubate at room temperature for 3 minutes. 
Centrifuge10000 x g, 3 min



SECTION: Genomic DNA Prep
10. Store genomic DNA at-20 °Cuntil library prep and sequencing.