In this Extraction I used: Invitrogen RNA mini kit, my Protocol, and the company manual from page 27.

Goal: Extract high quality RNA for sequencing

Sample Preparation

1. Add 550 uL trizol to a small frozen coral fragment in a 2 ml tube (if the sample is with RNA-safe, add equal volume of trizol)
2. Put samples into a cell disruptor for < 2 minutes at 2000 rpm
3. Transfer the liquid (~ 550 uL) into a new tube, add 450 uL trizol
4. Incubate 5 minutes at 15°-30° C
5. Add 100 uL BCP (1–bromo–3–chloropropane)
6. Vortex 15 seconds and incubate at 15°-30° C for 2-3 minutes
7. Centrifuge at 12000g for 15 minutes (in the meantime, prepare PureLink® DNase following step 14)
8. Transfer supernatant to a RNA-free tube and follow Invitrogen PureLink RNA Mini Kit (page 27 of the kit manual):
9. Add equal volume (~ 0.6 ml) of 70% ethanol to the tissue homogenate
10. Mix thoroughly by shaking or vortexing to disperse any visible precipitate that may form after adding ethanol
11. Transfer ≤700 μL of the sample (including any remaining precipitate) to the Spin Cartridge
12. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through, and reinsert the Spin Cartridge in the same Collection Tube
13. Repeat Steps 11–12 until the entire sample is processed. If DNA-free total RNA is required, proceed to On-column PureLink® DNase Treatment Protocol (page 63)
14. Prepare PureLink® DNase by adding the following components (supplied with PureLink® DNase) to a clean, RNase-free microcentrifuge tube. Prepare 80 μL per sample

1. Add 350 μL Wash Buffer I to the Spin Cartridge containing the bound RNA. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through and the Collection Tube. Place the Spin Cartridge into a new Collection Tube
2. Add 80 μL PureLink® DNase mixture (prepared as described above) directly onto the surface of the Spin Cartridge membrane
3. Incubate at room temperature for <15 minutes
4. Add 350 μL Wash Buffer I to the Spin Cartridge. Centrifuge at 12,000 x g for 15 seconds at room temperature. Discard flow-through and the Collection Tube and insert the Spin Cartridge into a new Collection Tube
5. Add 500 μL Wash Buffer II with ethanol (page 11) to the Spin Cartridge
6. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through, and reinsert the Spin Cartridge in the same Collection Tube
7. Repeat Steps 19-20 once
8. Centrifuge the Spin Cartridge at 12,000 × g for 1 minute at room temperature to dry the membrane with attached RNA. Discard the Collection Tube and insert the Spin Cartridge into a Recovery Tube
9. Add 100 μL RNase-Free Water to the center of the Spin Cartridge
10. Incubate at room temperature for 1 minute
11. Centrifuge for 2 minutes at ≥12,000 × g at room temperature
12. Proceed to analyzing RNA Yield and Quality (Nanodrop and TapeStation)