Goal: Metabolomic sample prep for Porites astreoides samples (adult and larvae)

Equipment needed

• 2mL dounces
• 11mm Snap Cap tubes
• 11mm Snap Cap auto sampler vials
• 100um cell strainer
• Methanol
• Acetonitrile
• Formic Acid
• Water

Make sure all equipment is autoclaved between each sample and to prepare reagents

Reagent Preparation

Extraction Buffer

• This extraction buffer is a ratio of 40:40:20 of methanol, acetonitrile, and water, with 0.5%[v/v] Formic Acid
• For a total of 200mL extraction buffer:
  • 80mL methanol
  • 80mL acetonitrile
  • 40mL water
  • 1mL formic acid
• Store at 4°C in a non-flammable fridge

15% Ammonium Bicarbonate Solution

• Solubility of Ammonium bicarbonate in water is: 220g/L for 100%
• 100% Ammonium Bicarbonate solution (250mL)
  • 55g dry Ammonium bicarbonate in 250mL LCMS water
• 15% Ammonium Bicarbonate Solution (250mL)
  • 37.5mL of 100% Ammonium Bicarbonate Solution + 212.5mL LCMS water
• Store at 4°C in a non-flammable fridge

Samples

• For this test, I am using multiple concentrations of larvae (8 samples) and adult tissue biopsies (4 samples). All samples were previously snap frozen and stored at -80°C.
  • Larvae are from a thermal stress experiment in 2017. All larvae were from the ambient treatment and from the patch reef.
  • Adults were from a histology experiment in 2018 and from 2 reef zones.

Table 1: Larval samples

Sample #	Vials	Total # of Larvae	Vial Labels
1	160	20	1-A, 1-B
2	230	20	2-A, 2-B
3	168,215	40	3-A, 3-B
4	174,208	40	4-A, 4-B
5	238,162,204	60	5-A, 5-B
6	251,172,210	60	6-A, 6-B
7	163,202,240,203	80	7-A, 7-B
8	167,217,253,270	80	8-A, 8-B

Table 2: Adult samples

Sample #	Coral ID	Vial (g)	Vial + Buffer (g)	Vial + Buffer + Coral (g)	Vial Labels
9	R13-20180731	1.1310	1.9755	2.3380	9-A, 9-B
10	R10-20180716	1.1260	1.9580	2.3268	10-A, 10-B
11	P6-20180716	1.1242	1.9520	2.2123	11-A, 11-B
12	P5-20180731	1.1362	1.9640	2.2952	12-A, 12-B

Preparation of samples before extraction

Larvae

• Thaw larvae on ice.
• Remove any residual seawater.
• Add 500uL of the extraction buffer to the vial and then transfer to the dounce.
• Add 500uL of the extraction buffer to the dounce and wait 5 minutes on ice.

Adult

• Label and weigh the vial empty, then with 1mL of the extraction buffer
• Using sterile clippers, clip approximately 0.5mm of coral tissue into the vial with 1mL of the extraction buffer.
• Weigh the vial with the extraction buffer and coral.
• Wait for 5 minutes on ice then transfer to dounce.

Extraction Protocol

• After waiting 5 minutes, homogenize the sample until the supernatant is visibly pigmented.
• Add an additional 500uL of cold extraction buffer to rinse the pestle.
• Strain the extract through a 100um cell strainer into a 50mL receptacle.
• Transfer extract to a new 1.5 mL tube.
• Vortex and centrifuge for 10 minutes at 15000g at 4°C.
• Remove 500uL of the supernatant to a new 1.5mL tube.
• Add 44uL of 15% ammonium bicarbonate.
• Vortex and spin down.
• Add 100uL into duplicate, labeled autosampler vials.
• Store 1.5mL tube and autosampler vials at -80°C.