Whole mussels stored in -80, took out one plastic bag with 5 mussels

Using Zymo Duet DNA RNA Extraction Kit HERE

1. Thawed bag on ice bucket until mussels could be opened
2. Started with smallest mussel first then went through to the largest

Sample	Location	Tide Pool	Date Collected
Mytilus 1	Bob Creek	TP5	8/13/2017
Mytilus 2	Bob Creek	TP5	8/13/2017
Mytilus 3	Bob Creek	TP5	8/13/2017
Mytilus 4	Bob Creek	TP5	8/13/2017
Mytilus 5	Bob Creek	TP5	8/13/2017

1. RNase Zap lab bench, forceps and scissors
2. For each mussel, prepped 5 1.5mL tubes. 4 with 750μl DNA/RNA Shield and 1 with 300μl DNA/RNA Shield
3. Open one mussel at a time and take biopsies from:
   • mantle (take two)
   • gill
   • heart
   • abductor muscle
4. Put one mantle tissue in the 300μl tube and the others in the 750μl tubes.
5. The 750μl tubes were labeled, put in the fridge for 24 hours then frozen at -80
6. RNase Zap bench and sterilize tools between each mussel
7. Added 30μl PK Digestion Buffer to each extraction tube (mantle tubes with 300μl)
8. Added 15μl Proteinase K to each extraction tube
9. Vortexed and spun down tubes
10. Put in Thermomixer at 55 degrees C shaking 1200
11. Samples not fully digested after 5 hours, let go overnight
12. Took samples out at 9:07am, still not fully digested
13. Spun down in centrifuge at max rcf for 2 minutes
14. Removed supernatant and put into a new 1.5mL tube
15. Proceeded with DNA/RNA Extraction protocol

DNA Extraction

1. Set up yellow DNA spin columns and collection tubes, label appropriately
2. Warm elution liquids to 70 degrees C (10mM Tris HCl pH. 8.0 and RNase free water)
3. Add equal volume (345µl) DNA/RNA lysis buffer to each sample tube
4. Finger flick to mix tubes
5. Add 700µl (total volume) of sample gently to the yellow DNA spin column
6. Centrifuge at 16,000 rcf (g) for 30 seconds
7. Important Save the flow through from this step: transfer to a new 1.5mL tube labeled for RNA
8. Add 400µl DNA/RNA Prep Buffer gently to the yellow DNA spin columns
9. Centrifuge at 16,000 rcf (g) for 30 seconds
10. Discard flow through (Zymo kit waste)
11. Add 700µl DNA/RNA Wash Buffer gently to the yellow DNA spin columns
12. Centrifuge at 16,000 rcf (g) for 30 seconds
13. Discard flow through (Zymo kit waste)
14. Add 400µl DNA/RNA Wash Buffer genetly to the yellow DNA spin columns
15. Centrifuge at 16,000 rcf (g) for 2 minutes
16. Discard flow through (Zymo kit waste)
17. Transfer yellow columns to new 1.5mL microcentrifuge tubes
18. Add 50µl warmed 10mM Tris HCl to each yellow DNA column by dripping slowly directly on the filer
19. Incubate at room temp for 5 minutes
20. Centrifuge at 16,000 rcf (g) for 30 seconds
21. Repeat last three steps for a final elution volume of 100µl
22. Label tubes, store at 4 degrees C if quantifying the same day or the next, if waiting longer store in -20

RNA Extraction

1. Add equal volume (700µl) 100% EtOH to the 1.5mL tubes labeled for RNA containing the original yellow column flow through
2. Vortex and spin down to mix
3. Add 700µl of that liquid to the green RNA spin columns
4. Centrifuge at 16,000 rcf (g) for 30 seconds
5. Discard flow through (Zymo kit waste)
6. Add 700µl to the green RNA spin columns (the rest from the 1.5mL RNA tubes)
7. Centrifuge at 16,000 rcf (g) for 30 seconds
8. Discard flow through (Zymo kit waste)
9. Add 400µl DNA/RNA Wash Buffer gently to each green RNA column
10. Centrifuge at 16,000 rcf (g) for 30 seconds
11. Discard flow through (Zymo kit waste)
12. Make DNase I treatment master mix:
    • 75µl DNA Digestion buffer x # of samples
    • 5µl DNase I x # of samples
13. Add 80µl DNase I treatment master mix directly to the filter of the green RNA columns
14. Incubate at room temp for 15 minutes
15. Add 400µl DNA/RNA Prep Buffer gently to each column
16. Centrifuge at 16,000 rcf (g) for 30 seconds
17. Discard flow through (Zymo kit waste)
18. Add 700µl DNA/RNA Wash Buffer gently to the yellow DNA spin columns
19. Centrifuge at 16,000 rcf (g) for 30 seconds
20. Discard flow through (Zymo kit waste)
21. Add 400µl DNA/RNA Wash Buffer genetly to the yellow DNA spin columns
22. Centrifuge at 16,000 rcf (g) for 2 minutes
23. Discard flow through (Zymo kit waste)
24. Transfer green columns to new 1.5mL microcentrifuge tubes
25. Add 50µl warmed DNase/RNase free water to each green RNA column by dripping slowly directly on the filer
26. Incubate at room temp for 5 minutes
27. Centrifuge at 16,000 rcf (g) for 30 seconds
28. Repeat last three steps for a final elution volume of 100µl
29. Label 1.5mL tubes on ice afterwards, and aliquot 5µl into PCR strip tubes to save for Qubit and Tape Station to avoid freeze-thaw of your stock sample
30. Store all tubes in the -80

Qubit

• Using Broad Range dsDNA and Broad Range RNA kits
• Protocol HERE
• All quantifications are in ng/µl
• All quantifications were taken twice

Sample	DNA Standard 1 (RFU)	DNA Standard 2 (RFU)	DNA 1 (ng/µl)	DNA 2 (ng/µl)	Average DNA	RNA Standard 1 (RFU)	RNA Standard 2 (RFU)	RNA 1 (ng/µl)	RNA 2 (ng/ul)	Average RNA
Mytilus 1	197	22011	47.2	45.6	46.4	426	11703	308	308	308
Mytilus 2	197	22011	49.2	48.4	48.8	426	11703	136	133	134
Mytilus 3	197	22011	191	187	189	426	11703	138	138	138
Mytilus 4	197	22011	354	350	352	426	11703	97.2	96.6	96.9
Mytilus 5	197	22011	112	110	111	426	11703	884	880	882

Tape Station DNA and RNA

Protocol HERE and

Links to tapes:

RNA

DNA

RNA is very degraded