PROTOCOL: DNA/RNA Extractions of P. generosa samples

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This protocol was recently revised for testing geoduck samples (link 1 and link 2) modified from successful extractions of coral tissue link here.

Zymo Research Quick-DNA/RNA MiniPrep Plus Kit - link to this kit here

Prep lab space

Set your space with the following…

N = the number of samples you are running today

• N × 1
  • yellow spin columns (DNA)
  • green spin columns (RNA)
    • both yellow and green spin columns inserted in flow-through tubes
    • lid == sample ID and either ‘D’ or ‘R’
• N × 2
  • 1.5 ml epindorf tubes labelled for (1) DNA and (2) RNA;
    • lid == sample ID and either ‘D’ or ‘R’
    • tube == sample ID, ‘DNA’ or ‘RNA’, date of extraction, and initials (i.e. 852 DNA 20200818 SG)
  • 5 ml tubes labelled for (1) DNA and (2) RNA
    • lid == sample ID and either ‘D’ or ‘R’
• N / 2
  • glass microbead vials
    • Note: each microbead vial contains ~0.5 ml and 0.25 ml is used to homogenize each geoduck sample

• Zymo waste container (i.e. labelled glass beaker, 50 ml)

• Warm the elution liquids to 70 °C for the DNA (10 mM Tris) and RNA extractions (RNAse free water)
  • How much 10mM Tris?
    • N × 100 µl
  • How much RNase free water?
    • N × 100 µl
  • Use a Thermomixer to keep the two samples warm

Geoduck Homogenization

1. Remove samples from -80 °C - samples are whole juvenile geoduck with one animal per 1.5 ml screw cap tube.
2. Add 1 mL of RNA/DNA shield to each
3. Add 0.25 mL of 0.5mm glass beads to each tube (add to the same tube from the -80 °C)
4. Vortex for one minute. Leave the settings on and at mid-max power.
   • Important: The one minute at max power is only for large individuals of >8mm shell length - these individuals are tightly fit into the 1.5 ml screw cap tubes. Smaller indivuals (~ 3- 5mm shell length) should be vortexed for a shorter duration or lower power.
   • Note: must insert the tubes firmly in the vortex or they will fly out. Hover a hand lightly over the samples.
5. Spin down all tubes to reduce the bubbles and move large tissue fragments and/or suspended glass beads to the bottom of the vial
6. Observe the samples - Is the supernatant clear without large fragments of suspended tissue?

• If yes, proceed to step 8

• If no, proceed to step 7

1. You answered no the #6 meaning supernatant transfer is not possible without transfering suspended tissue fragment(s). Spin this tube down again and observe, if it is possible to take the supernantant (~700ul) without transferring tissue proceed to step 8. Alternatively, take what you can of this tube and transfer to a new labelled 1.5 ml epindorf tube - spin the tube down. Now you should be able to proceed to step 8.

2. In a new labelled 1.5 ml tube, add the following:
   • supernatant of sample
   • i.e. 700 ul - Proteinase K digestion buffer: add 1:10 of Proteinase K digestion buffer : supernatant
   • i.e. add 70 ul Proteinase K digestion buffer if you previously added 700 ul of supernatant - Proteinase K: add 1:2 of Proteinase K : Proteinase K digestion buffer
     • i.e. add 35 ul Proteinase K if you previously added 70 ul Proteinase K digestion buffer
3. Vortex and spin down all tubes.
4. Transfer supernatant to a new 5 mL tube.
   • Important: this volume depends on the volume taken in step #8
   • i.e. if you added 700 ul of supernatant in step 8 (along with proteinase reagents), you will be able to transfer 800 ul here in step 10.

Extraction: Zymo Research Quick-DNA/RNA MiniPrep Plus Kit

DNA Extraction of Geoduck Samples

1. Set up yellow DNA spin columns and collection tubes, label appropriately
   • N = 1 yellow spin column per sample
2. Add DNA/RNA lysis buffer in equal volume to supernatant (in 5ml tube)
   • i.e. 800 ul DNA/RNA lysis buffer added if 800 ul were transfered in step #8 in ‘Geoduck Homogenization’

3. Finger flick to mix tubes

4. Add half of the total volume of sample gently to the yellow DNA spin column

4a. Centrifuge at 16,000 rcf (g) for 30 seconds

4b. SAVE THE FLOW THROUGH for RNA extractions! from this step: transfer to a new 5 mL tube labeled for RNA extractions

NOTE: Repeat steps 5-7 until all volume is gone. (i.e. x 2)

• i.e. if total volume was 1600 µl, you would run steps 5-7 twice with 800 µl each run

• record the total volume of you transfered for RNA!

1. Add 400 µl DNA/RNA Prep Buffer gently to the yellow DNA spin columns

5a. Centrifuge at 16,000 rcf (g) for 30 seconds

5b. Discard flow through (Zymo kit waste)

1. Add 700 µl DNA/RNA Wash Buffer gently to the yellow DNA spin columns

6a. Centrifuge at 16,000 rcf (g) for 30 seconds

6b. Discard flow through (Zymo kit waste)

1. Add 400 µl DNA/RNA Wash Buffer genetly to the yellow DNA spin columns

7a. Centrifuge at 16,000 rcf (g) for 2 minutes

7b. Discard flow through (Zymo kit waste)

1. Transfer yellow columns to new 1.5 mL microcentrifuge tubes

2. Add 50 µl warmed 10mM Tris HCl to each yellow DNA column by dripping slowly directly on the filter

9a. Incubate at room temperature for 5 minutes. Centrifuge at 16,000 rcf (g) for 30 seconds keep the flow through

1. Add another 50 µl warmed 10mM Tris HCl to each yellow DNA column by dripping slowly directly on the filter

10a. Incubate at room temperature for 15 minutes. Centrifuge at 16,000 rcf (g) for 30 seconds. Throw away filter and keep flow through.

1. For QC… Aliquot 10 µl of the elution to 0.5 mL PCR tubes for Qubit and Gel Electrophoresis analysis.
   • N = 1 tube per sample
2. Storage:
   • QC aliquots: store at 4 °C if quantifying the same day or the next
   • Whole extractions and QC aliquots: store at -20 °C if waiting longer

RNA Extraction of Geoduck Samples

1. Add equal volume 100% EtOH to the tubes labeled for RNA containing the original yellow column flow through
   • i.e. 1600 ul 100% EtOH added if 1600 ul were transfered in step #4 in ‘DNA Extractionof Geoduck Samples’
   • Note: Review # 4 in ‘DNA Extraction’. You should have the total volume that you transferred from the spin column in this step.

2. Vortex and spin down to mix

3. Add 1/4 of the total volume to the green RNA spin columns
   • i.e. if the total volume was 1600 ul then add 800 ul here

3a. Centrifuge at 16,000 rcf (g) for 30 seconds

3b. Discard flow through (Zymo kit waste)

1. Repeat step 3 until all volume is gone (i.e. x 4)
   • Note: get the DNase I from the -20°C
2. Add 400 µl DNA/RNA Wash Buffer gently to each green RNA column

5a. Centrifuge at 16,000 rcf (g) for 30 seconds

5b. Discard flow through (Zymo kit waste)

1. Make DNase I treatment master mix:
   • N × 75µl DNA Digestion buffer
   • N × 5µl DNase I

N	DNA Digestion buffer	DNase I
1	75	5
3	225	15
5	375	25
10	750	50

1. Add 80 µl DNase I treatment master mix directly to the filter of the green RNA columns

7a. Incubate at room temp for 15 minutes

1. Add 400 µl DNA/RNA Prep Buffer gently to each column

8a. Centrifuge at 16,000 rcf (g) for 30 seconds

8b. Discard flow through (Zymo kit waste)

1. Add 700 µl DNA/RNA Wash Buffer gently to the green RNA spin columns

9a Centrifuge at 16,000 rcf (g) for 30 seconds

9b. Discard flow through (Zymo kit waste)

1. Add 400 µl DNA/RNA Wash Buffer genetly to the green RNA spin columns

10a. Centrifuge at 16,000 rcf (g) for 2 minutes

10b. Discard flow through (Zymo kit waste)

1. Transfer green columns to new 1.5 mL microcentrifuge tubes

2. Add 50 µl warmed DNase/RNase free water to each green RNA column by dripping slowly directly on the filter

13a. Incubate at room temp for 5 minutes

13b. Centrifuge at 16,000 rcf (g) for 30 seconds

1. Repeat step 18 for a final elution volume of 100 µl

2. For QC… Aliquot 5 µl of the final elution to 0.5 mL PCR tubes for Qubit and TapeStation analysis.
   • N = 1 tube per sample
3. Storage:
   • QC aliquots: store on a bead bucket if measuring same day
   • Whole extractions and QC aliquots: store 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.