Goal

To test the protocol for DNA/RNA extractions for airbrushed homogenates of Porites astreoides for the Thermal Transplant 2017/2018 experiment.

Samples

I used 6 adult P. astreoides samples that were snap frozen, then airbrushed with filtered seawater. 0.5 mL of the homogenate was aliquoted then re-frozen at -80 °C. I used 3 corals from two different time points of the experiment, post treatment (2017) and post-transplant (2018)

Sample Numbers

• 17-26
  • P12-T2 (2017: Patch + High Treatment)
• 17-295
  • R7-T2 (2017: Rim + Ambient Treatment)
• 17-682
  • R19-T2 (2017: Rim + High Treatment)
• 18-19
  • R7-B (2018: Rim + Ambient Treatment + Rim Transplant)
• 18-31
  • P12-B (2018: Patch + High Treatment + Rim Transplant)
• 18-43
  • R19-B (2018: Patch + High Treatment + Rim Transplant)

Protocol description

I used a modified version of this protocol for homogenates.

Homogenate preparation

1. Immediately after taking sample out of the -80°C freezer, add 500μL of DNA/RNA shield
2. Once fully thawed, add 100 μL of Protenase K digestion buffer (Derived from volume of homogenate and 300/30 ratio from Zymo Protocol) and 50 μL of Protenase K (Derived from volume of homogenate and 300/15 ratio from Zymo Protocol)
3. Vortex and spin down.
4. Incubate on Thermomixer at 56°C at 1500 rpm for 30 minutes
5. Spin down for 30 seconds to pellet any debris
6. Remove 1000 μL of the supernatant and transfer in 5 mL tubes. Avoid any debris.

Zymo Duet RNA DNA Extractions

Modified from the Zymo protocol.

DNA Extraction

1. Add equal volume of DNA lysis buffer (1000 μL) as sample volume to the 5 mL tube with sample
   • Vortex to mix
2. Transfer 700 μL into DNA filter column (yellow)
   • Centrifuge at 16,000 rcf for 30 seconds
   • Remove flow through liquid and transfer into a new 5 mL tube labeled for RNA
3. Repeat steps 2 until all liquid is gone (x 3)
4. Add 400 μL of DNA/RNA prep buffer to column
   • Centrifuge at 16,000 rcf for 30 seconds
   • Remove the flow through and transfer to waste
5. Add 700 μL of DNA/RNA wash buffer
   • Centrifuge at 16,000 rcf for 30 seconds
   • Remove the flow through and transfer to waste
6. Add 400 μL of DNA/RNA wash buffer
   • Centrifuge at 16,000 rcf for 2 minutes
   • Remove the flow through and transfer to waste
7. Removed column and place into a new sterile 1.5 mL tube
8. Add 50 μL of 10 mM Tris HCL (warmed to 55°C) directly to filter
   • Incubate at room temperature for 15 minutes
   • Centrifuge at 16,000 rcf for 30 seconds
   • Keep flow through in tube
9. Add another 50 μL of 10 mM Tris HCL (warmed to 55°C) directly to filter
   • Incubate at room temperature for 5 minutes
   • Centrifuge at 16,000 rcf for 30 seconds
   • Discard column and keep flow through in tube
10. Aliquot 10 μL into PCR tubes for QC.
11. Store remaining aliquot at -20°C

RNA Extraction

1. Add equal volume of 100% Ethanol (2000 μL) as sample volume to the 5 mL tube with sample
   • Vortex to mix
2. Transfer 700 μL into RNA filter column (green)
   • Centrifuge at 16,000 rcf for 30 seconds
   • Remove flow through liquid and transfer into a new 5 mL tube labeled for Protein
3. Repeat steps 12 until all liquid is gone
4. Add 400 μL of DNA/RNA wash buffer to column
   • Centrifuge at 16,000 rcf for 30 seconds
   • Remove the flow through and transfer to waste
5. Make DNase I master mix: [75μL DNA digestion buffer and 5μL DNase] X n (sample #)

   Today I had 6 samples therefore: 450 μL of DNA digestion buffer and 30 μL of DNase I

6. Add 80μL of DNase I master mix directly to the filter of each column. Incubate at room temperature for ~15 minutes
7. Add 400 μL of DNA/RNA prep buffer to column
   • Centrifuge at 16,000 rcf for 30 seconds
   • Remove the flow through and transfer to waste
8. Add 700 μL of DNA/RNA wash buffer
   • Centrifuge at 16,000 rcf for 30 seconds
   • Remove the flow through and transfer to waste
9. Add 400 μL of DNA/RNA wash buffer
   • Centrifuge at 16,000 rcf for 2 minutes
   • Remove the flow through and transfer to waste
10. Removed column and place into a new sterile 1.5 mL tube
11. Add 50 μL of RNAase-free water (warmed to 55°C) directly to filter
    • Incubate at room temperature for 5 minutes
    • Centrifuge at 16,000 rcf for 30 seconds
    • Keep flow through in tube
    • Made the gel during this incubation
12. Add another 50 μL of RNAase-free water (warmed to 55°C) directly to filter
    • Incubate at room temperature for 5 minutes
    • Centrifuge at 16,000 rcf for 30 seconds
    • Discard column and keep flow through in tube
    • Took out qubit standards
13. Aliquot 5 μL in PCR strip tubes for QC.
14. Store remaining aliquot at -80°C

Clean-up

1. Place tissue and liquid in the waste container labeled Zymo extraction waste.
2. Wipe down RNA free area with RNase away and kimwipes.
3. Throw away all tips and restock tip boxes if necessary.

Testing Quantity and Quality

Quantify Results

Qubit

To test RNA and DNA quantity: Qubit

6 samples + 2 standards + 1 error = 9

DNA Broad Range

199 µl Buffer x 9 = 1791 µl Buffer 1 µl Reagent x 9 = 9 µl Reagent

RNA Broad Range

199 µl Buffer x 9 = 1791 µl Buffer 1 µl Reagent x 9 = 9 µl Reagent

Sample	DNA (ng/uL)	RNA (ng/uL)
Standard 1	201.52	396.07
Standard 2	21429.55	9327.86
17-26	10.3	24.8
17-295	21.6	34.6
17-682	22.8	28.6
18-19	16.2	30.2
18-31	6.36	10.0
18-43	17.0	22.6

Gel Electrophoresis

To test DNA quality: Gel Electrophoresis

• Today I did a small gel of the above protocol and ran 6 samples for MS

TapeStation

To test RNA quality: TapeStation

Overall Summary:

Sample #17-26:

Sample #17-295:

Sample #17-682:

Sample #18-19:

Sample #18-31:

Sample #18-43: