TITLE: RNA extraction for Karlodinium veneficum PLY720
AUTHORS: Will Lewis

[DESCRIPTION]
A methodology for cell harvesting and RNA extraction from Karlodinium veneficum for the purposes of RNA-seq.

[BEFORE_START]
Cool centrifuges to 4°C.

[GUIDELINES]
Clean all pipettes and bench with ethanol and then RNase Zap before processing the collected cell pellets.

Regularly change gloves and spray with RNase Zap.

Ideally use newly opened boxes of filter tips.

Adapted from the TRIzol protocol available on the ThermoFisher website: https://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf

[STEPS]
SECTION: Cell harvesting
1.  
Grow 200 ml of Karlodinium veneficum PLY720 culture in L1 medium. Once cultures have grown to a density between 50,000 and 300,000 cells/ml (corresponding to exponential growth phase) they can be harvested.

SECTION: Cell harvesting
2. For each culture being processed, gently invert the culture flask several times to homogenise the culture and then transfer four 50 ml aliquots of the culture to four 50ml centrifuge tubes. 
SECTION: Cell harvesting
4. Once the pellets are resuspended they need to be pooled by combining them using a 1000 µl pipette and transfering them to a 2 ml microcentrifuge tube.
SECTION: Cell lysis and phase separation
7. 
Remove cells pellets from -80 °C storage and then process immediately. Work in a fume hood from this point forward whenever a tube is open. for each cell pellet, resuspend in 1 ml TRIzol reagent using a 1 ml pipette and pipette up and down to homogenise. This will lyse the cells and in doing so release their pigments, meaning that the mixture will be brown in colour.
SECTION: Cell lysis and phase separation
8. Incubate the tube for 5 minutes at room temperature.
SECTION: Cell lysis and phase separation
9. Add 200 µl chloroform to the tube and seal again.
SECTION: Cell lysis and phase separation
10. Gently shake the tube to mix the chloroform with the lysed cells in TRIzol and then incubate at room temperature for 2-3 minutes.
SECTION: Cell lysis and phase separation
11. Centrifuge all samples for 15 minutes at 12000 x g at 4°C.

The samples should separate into a lower phase, an interphase and an upper aqueous phase. Due to the presence of the brown colour pigments, the boundary between the upper aqueous phase and interphase might be slightly more difficult to see than for a non-pigmented organism, and therefore has to be recognised by a change in turbidity and shade, as opposed to a change in colour.
SECTION: Cell lysis and phase separation
12. Using a 200 µl pipette, carefully transfer the upper aqueous phase containing the RNA to a sterile RNase-free 1.5 ml tube for each sample. Be conservative in doing this and leave some of the upper phase behind so as not to risk making contact with the DNA-containing interphase, thereby trying to minimise carry-over of DNA. Do not touch the sides of the tube with the pipette tip.
SECTION: RNA precipitation
13. Add 0.5 ml isopropanol to the upper aqueous phase and incubate for 10 minutes at room temperature.
SECTION: RNA precipitation
14. Centrifuge the sample at 12,000 x g for 10 minutes at 4°C. The precipitated RNA should form a white or gel-like pellet at the bottom of the tube.
SECTION: RNA precipitation
15. Remove and discard the supernatant using a 1 ml pipette for each sample.
SECTION: Wash the RNA
16. Resuspend each pellet in 75% ethanol. The pellet does not need to be broken-up by vigorous pipetting or vortexing, just dislodge the pellet from the bottom of the tube by pipetting up and down with the ethanol.
SECTION: Wash the RNA
17. Centrifuge the resuspended RNA pellet samples at 7500 x g at 4°C.
SECTION: Wash the RNA
18. Remove the supernatant with a 1 ml pipette.
SECTION: Wash the RNA
19. Repeat the previous three steps at least a further two times, thereby washing the RNA pellet in total three times in 75% ehtnaol. This seems to reduce the amount of phenol and other contaminant carry-over, improving 260/280 and 260/230 ratios measured later with Nanodrop.
SECTION: Wash the RNA
20. After the final wash remove as much of the supernatant as possible, first removing the majority with a 1ml pipette, then the remainder with a 10 µl pipette.
SECTION: Dissolve the RNA
21. Briefly centrifuge the tube to collect any remaining liquid and again remove this with a 10µl pipette. Repeat this once more to ensure all liquid is removed and the pellet is dry before immediately adding 100µl UltraPure™ DNase/RNase-Free Distilled Water to each sample. If the pellet stays dry for too long it may not redissolve. Pipette the water gently up and down to dislodge pellet from the bottom of the tube.


SECTION: Dissolve the RNA
22. Place the tube containing the resuspended RNA pellet and water in a heat block set to 57 °C for 10 minutes
SECTION: Dissolve the RNA
23. Visually confirm whether the RNA has dissolved. Gentle pipetting up and down may be required to see whether the RNA has dissolved fully and to ensure the sample is mixed well prior to quantitifcation. If the RNA concentration in the samples is high, then samples may need to be incubated for longer or another 50-100µl UltraPure™ DNase/RNase-Free Distilled Water added.
SECTION: Quantification, quality control and storage
24. Aliquot each sample, label and store at -80°C. I normally aliquot each sample in at least three tubes. This means that you do not need to thaw and then refreeze the entire sample, potentially compromising RNA integrity, and instead just thaw a single sample to perform quantification and other quality control analyses.
SECTION: Quantification, quality control and storage
25. Analyse samples using Nanodrop to ensure the 260/280 ratio is ~2.0 and the 260/230 ratio is between 2.0 and 2.2.
SECTION: Quantification, quality control and storage
26. Measure concentration of RNA and DNA using Qubit BR assay kits.
SECTION: Cell harvesting
3. Centrifuge the 50 ml tubes at 5000 x g for 5 minutes at 4°C. Decant and dispose of the supernatants from each tube by pouring, leaving the pellet in the bottom of the tubes. Inverting the tube in one smooth hand motion to remove the supernatant removes the majority of the liquid but should leave a small volume of liquid on the walls of the tubes. This is desirable as then once you reinvert the tube, the liquid will run back down to the pellet. This small volume of remaining liquid, which typically is approximately 500 µl in volume, can then be used to resuspend the pellet using a pipette.
SECTION: Cell harvesting
5. Centrifuge the microcentrifuge tube at 5000 x g for 5 minutes at 4°C and then dispose of the supernatant using a 1000 µl pipette.
SECTION: Cell harvesting
6. Flash-freeze the cell pellets by sealing the tubes and dropping them into a liquid N2 bath for several minutes. The tubes can then be removed and stored at -80 °C until further processing for RNA extraction.