TITLE: Virus and Prokaryote Enrichment in Coral DNA Metagenomes
AUTHORS: Natascha Varona, Bailey Wallace, Cynthia Silveira

[DESCRIPTION]
Tissue preparation and DNA extraction protocol for enrichment of viruses and prokaryotes in DNA metagenomes of corals.

[BEFORE_START]
Glass beads must be washed with 0.1 N HCl
Incubator set to 56 °C & 70 °C
Check if Buffer B5 and Proteinase K were prepared according to section 3 of NucleoSpinⓇ Tissue Kit Manual.



[STEPS]
SECTION: Sample Preparation
1. Collect a coral sample of approximately 1cm3 containing coral tissue, mucus, and skeleton. Use a fresh sample if possible. If not, flash-freeze the sample and keep it at  -80 °C until processing. Thaw the sample on ice before proceeding.

SECTION: Sample Preparation
2. Weigh and record coral fragment weight.
SECTION: Sample Preparation
3. Crush the coral sample using a sterile mortar and pestle until the skeleton and tissue fragments are the size of coarse sand. Do not overdo this step. Transfer the fragments, along with the mucus (coral homogenate), to a 2 mL microcentrifuge tube.
SECTION: Sample Preparation
4. Suspend sample in 100 µL of Artificial Seawater (ASW) and 0.2 g of sterile glass beads.

SECTION: Sample Preparation
5. Vortex at speed 3 for 5 min to dislodge tissue and mucus.
* To avoid lysis of bacterial cells, do not vortex the sample at higher speeds or longer durations than described.
SECTION: Sample Preparation
6. Pulse-centrifuge the sample (3-5 seconds at 3.200 x g).

SECTION: Sample Preparation
7. Collect supernatant and transfer to a clean 15 mL microcentrifuge tube.

SECTION: Sample Preparation
8. Bring sample up to 1 mL with sterile ASW.

SECTION: Sample Preparation
9. Add 100 µL of 10X DNase Buffer and 2 µL of DNase 10,000 U/mL for a final concentration of 20 U/mL. Mix by gently inverting the tube.

SECTION: Sample Preparation
10. Incubate at room temperature for 120 min .

SECTION: Sample Preparation
11. Stop DNase activity by adding 10 µL of 100X EDTA.
SECTION: Sample Preparation
12. Bring sample volume up to 5 mL with sterile ASW.

SECTION: Sample Preparation
13. Transfer thesample to a 10 mL syringe and push through an 8.0 µm, 25 mm flat filter, collecting the flowthrough in a clean 15 mL centrifuge tube.
* If the sample clogs the filter, replacing the 8.0 µm filter may be necessary. In this case, pull back on the syringe plunger to recollect the sample in the syringe before transferring it to a new filter. 
* In the case of samples with excessive mucus, you may further dilute the sample with sterile ASW prior to filtering.
SECTION: Sample Preparation
14. Transfer flowthrough to an Amicon‱ Ultra-15 Centrifugal Filter Unit.
SECTION: DNA Extraction
16. Add 200 µL Buffer T1 and 20 µL of Proteinase K from the NucleoSpinⓇ Tissue Kit to the Amicon‱ filter unit.

SECTION: DNA Extraction
17. Rinse one side of the filter with the lysis buffer 3-5 times using a pipette.
SECTION: DNA Extraction
18. Securely close the Amicon‱ filter unit and incubate for 60 min at 56 °C, being sure to place the tube in a position where the rinsed side of the filter remains covered in the Buffer T1 & Proteinase K mixture.
* Some Amicon‱ filter unit lids may not seal properly. Check for leaks before placing in the incubator. Seal with parafilm if necessary.

SECTION: DNA Extraction
19. Repeat steps 17 and 18 on the other side of the filter.
SECTION: DNA Extraction
20. Add 210 µL Buffer B3 to the Amicon‱ filter unit and vortex to mix.

SECTION: DNA Extraction
21. Incubate for 10 min at 70 °C .

SECTION: DNA Extraction
22. Add 210 µL ethanol (100%) to the Amicon‱ filter unit and vortex to mix.

SECTION: DNA Extraction
23. Proceed with steps 5-7 of the NucleoSpinⓇ Tissue Kit Standard protocol for human or animal tissue and cultured cells.
SECTION: DNA Extraction
24. On step 8 of the NucleoSpinⓇ Tissue Kit, perform the following:
a. Place the NucleoSpinⓇ Tissue Column into a 1.5 mL microcentrifuge tube (not provided in kit)
b. Add 50 µL PCR-grade water
c. Incubate at room temperature for 1 min 
d. Centrifuge for 1 min at 11000 x g
e. Repeat steps b, c, & d once, for a final volume of 100 uL

SECTION: Sample Preparation
15. Centrifuge at room temperature for 30 min at ≥2600 x g.