TITLE: Labyrinthulomycete total RNA extraction protocol - hot phenol
AUTHORS: Jackie Collier

[DESCRIPTION]
Modified from Lippmeier et al. 2009; developed as part of the labyrinthulomycete JGI Community Sequencing Project and Gordon and Betty Moore Foundation Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP)

[STEPS]
SECTION: Preparing biomass and reagents
1. Grow up cells, collect, and freeze rapidly - preferably in liquid nitrogen. Store biomass at -80C if not extracting immediately. 
This protocol has worked so far for two different thraustochytrids (Aurantiochytrium limacinum ATCC MYA-1381, Schizochytrium aggregatum ATCC 28920) and two Aplanochytrium strains (PBS06 and PBS07). 
SECTION: Preparing biomass and reagents
2. Prepare extraction buffer 
100 mM Tris-HCl pH 7.5
1.5 M NaCl
50 mM Na2-EDTA pH 8.0
20 g per liter CTAB (cetyltrimethylammonium bromide)
8 mg per ml DTT (dithiothreitol) - ADD FRESH JUST BEFORE USE
(note, if you keep these stocks in glass bottles, first soak the bottles with 0.1 M NaOH to inactivate RNAses)

Prepare Tris-equilibrated phenol, pH 4.5-4.8, by warming to 65 C
Prepare acid phenol:chloroform:isoamyl alcohol (pH 4.5-4.8, 125:24:1) by warming to 65 C
Set up to incubate the extraction step at 65 C

Prepare 8M LiCl

Chill 100% ethanol and 70% ethanol

Prepare TE or nuclease-free water

Get a bucket of ice

SECTION: Extraction steps - REPEAT 3 TIMES (do 3 extractions)
3. Suspend ~1000 micrograms wet weight biomass per ~10 ml extraction buffer by vortexing
(these amounts would yield tens of micrograms of total RNA)
SECTION: Precipitation steps
8. Add an equal volume of isopropanol and incubate overnight at 4 C
SECTION: Extraction steps - REPEAT 3 TIMES (do 3 extractions)
3.1. Incubate at 65 C for 5 minutes, mix by vortex or inverting 2 or 3 times
SECTION: Extraction steps - REPEAT 3 TIMES (do 3 extractions)
4. Add equal volume hot (65 C) acid phenol/chloroform/isoamyl alcohol, votes to mix well
SECTION: Extraction steps - REPEAT 3 TIMES (do 3 extractions)
4.1. Incubate at 65 C for 5 minutes, mix by vortex or inverting 2 or 3 times
SECTION: Extraction steps - REPEAT 3 TIMES (do 3 extractions)
5. Cool the mixture in ice
SECTION: Extraction steps - REPEAT 3 TIMES (do 3 extractions)
6. Centrifuge to separate the phases 4 C, 6,000g, 15 minutes
SECTION: Extraction steps - REPEAT 3 TIMES (do 3 extractions)
7. Move the top phase to a new tube, avoiding the interface
SECTION: Precipitation steps
8.1. If remaining DNA forms a large fluffy precipitate, remove by spooling onto glass rod
SECTION: Precipitation steps
9. Collect precipitate by centrifugation 4 C, 16,000g, 30 minutes
(may require distributing the sample among several 1.5 ml microfuge tubes and/or multiple spins for each tube to process the volume)
SECTION: Precipitation steps
10. Wash each pellet with 0.5 ml ice-cold 75% ethanol twice, centrifuging 4 C 16,000g, 5 minutes
SECTION: Precipitation steps
10.1. Allow the pellets to air dry
SECTION: Precipitation steps
11. Dissolve pellets in an appropriate volume, analyze by appropriate method (spectrophotometry, Bioanalyzer); store frozen at -80 C
SECTION: Optional cleanup steps
12. If the RNA contains particulates that do not dissolve, they can be removed by centrifugation 4 C, 6,000g, 5 minutes and transferring the supernatant to a fresh tube.
SECTION: Optional cleanup steps
14. If the RNA contains large amounts of DNA or other contaminants, it can be removed by selectively precipitating the RNA with 17 microliters of 8M LiCl per 50 microliters RNA
SECTION: Optional cleanup steps
14.1. Incubate -20 C 60 minutes
SECTION: Optional cleanup steps
14.2. Collect RNA by centrifugation 4 C, 16,000g, 20 minutes
SECTION: Optional cleanup steps
15. Wash each pellet with 0.5 ml ice-cold 75% ethanol twice, centrifuging 4 C 16,000g, 5 minutes
SECTION: Optional cleanup steps
15.1. Allow the pellets to air dry
SECTION: Optional cleanup steps
16. Dissolve pellets in an appropriate volume, analyze by appropriate method (spectrophotometry, Bioanalyzer); store frozen at -80 C
SECTION: Optional cleanup steps
13. Even if the RNA does not have obvious DNA, a DNAse treatment step is nonetheless recommended