TITLE: Isolation and RNA extraction from Marine Protist Single Cells for Transcriptomics (sc-RNAseq)
AUTHORS: Sarah Romac

[DESCRIPTION]
It is difficult to get clean RNA from uncultivated protists (phyto and zooplankton), as they have to be isolated by micromanipulation and very sensitive to exogenous contaminations.
Here we optimized an extraction protocol to get clean DNA-free RNA from protist single-cells for transcriptomic studies.



[BEFORE_START]
Prepare Ethanol 70% from absolute Ethanol : Mix 35 mL of absolute Ethanol with 15 mL nuclease-free water in a Falcon 50mL Store at -20°C.

[STEPS]
SECTION: 1.    Cell Isolation
1. Isolate individually protist cells (5-500µm in length) using a glass bent micropipette (or a micropipette adapted to the length of the microorganism) under a binocular microscope or an inverted microscope. 

SECTION: 1.    Cell Isolation
2. Wash each cell in three successive baths of 0.22µm-filtered and sterile seawater on a glass slide. 

Wash glass bent in H2O Nuclease-free water, and EtOH96%, and then filtered seawter <0,2µm between each bath.
Image the isolated cell.
SECTION: 1.    Cell Isolation
3. Add 30 μL of lysis buffer (Tissue and Cell Lysis Solution from MasterPureTM Complete DNA and RNA Purification Kit, Epicenter) in a 1.5mL sterile microtube.

SECTION: 1.    Cell Isolation
4. Transfer subsequently single-cell in the 1.5mL microtube, flash-freeze in liquid Nitrogen and store at -80°C for RNA/DNA extraction.
SECTION: 2.	Cell lysis
5. Pellet cells by short-spin centrifugation.
SECTION: 2.	Cell lysis
6. Dilute 1 µL of Proteinase K in 300 µL de lysis solution Tissue et Cellule for each sample. Vortex 10 sec for resuspending cells (facultative).
SECTION: 2.	Cell lysis
7. Add 300 µL of mix Proteinase K + lysis solution Tissue et Cellule in each sample. Vortex.
SECTION: 2.	Cell lysis
8. Incubate 15 min at 65°C , 600 rpm. Put samples in ice 3-5 min.
SECTION: 3.	Total nucleic acids precipitation
9. Add 150 µL MPC reagent to 300 µL of lysed sample. Vortex.
SECTION: 3.	Total nucleic acids precipitation
10. Spin 10 min at 11 000 g, 4°C. If there is no pellet, add more 25 µL MPC buffer and spin again 10 min at 11000 g, 4°C.
SECTION: 3.	Total nucleic acids precipitation
11. Transfer the supernatant ion a new clean microtube (1,5 mL), discard the pellet.
(To keep the squeletton, keep the tube with the pellet, add 500 µL MilliQ Water and store at -20°C).
SECTION: 3.	Total nucleic acids precipitation
12. Add 500 µL of Isopropanol. Mix per inversion. Spin 10min at Vmax (~20 000 g), 4°C.
SECTION: 3.	Total nucleic acids precipitation
13. Discard the supernatant with precaution, without touching the pellet.
SECTION: 3.	Total nucleic acids precipitation
14. Add 500 µL of Ethanol 70%. Don’t vortex, mix gently the support. Spin 5min at Vmax (~20 000 g), 4°C.
SECTION: 3.	Total nucleic acids precipitation
15. Discard a maximum of supernatant with precaution using both P1000 and P100, without touching the pellet.
SECTION: 3.	Total nucleic acids precipitation
16. Let dry 5-10 min at room temperature until the pellet becomes transparent.
SECTION: 3.	Total nucleic acids precipitation
17. Elute in 33 µL of TE1x buffer. Vortex and spin shortly.
1- RNA : Take an aliquot of  30 µL and transfer it in a 0.2mL
sterile microtube. Proceed immediately to the TurboDNase treatment.
2- DNA : You should have 3µL left for barcoding (PCR 18S1, 18S2, 28S). Store at -20°C.
SECTION: 4. TurboDNase treatment (Work on ice)
18. To 30µL of each RNA extract, add :
- 3 µL TurboDNase buffer
- 1µL (2u) of TurboDNase
Do a molecular flick.

Incubate 25min at 37°C in the thermocycleur.
Replace on ice and let incubate on ice for 2min. 
SECTION: 4. TurboDNase treatment (Work on ice)
19. Add again 1µL (2u) of TurboDNase.
Do a molecular flick.
Incubate 25min at 37°C in the thermocycleur.
Replace samples on ice. Let incubate on ice for 2min and proceed immediately to RNA concentration.

SECTION: 5. RNA purification and concentration using RNA Clean and Concentrator-5 kit (ZymoResearch)
20. Adjust volume of RNA extract to 50µL (Add 15 µL TE1x MP buffer to the 35µL of TurboDNase product).
SECTION: 5. RNA purification and concentration using RNA Clean and Concentrator-5 kit (ZymoResearch)
21. Add 2v (100µL) of RNA binding buffer. Mix thoroughly.
Add 1v (150µL) of EtOH 96-100%. Mix thoroughly.

SECTION: 5. RNA purification and concentration using RNA Clean and Concentrator-5 kit (ZymoResearch)
22. Transfer into a Zymo-Spin IC Column in a Collection Tube.
Centrifuge 30sec at 11 000g, at room temperature.
SECTION: 5. RNA purification and concentration using RNA Clean and Concentrator-5 kit (ZymoResearch)
24. Add 700 µL RNA Wash Buffer. Centrifuge 2min at 11 000g.
SECTION: 5. RNA purification and concentration using RNA Clean and Concentrator-5 kit (ZymoResearch)
23. Add 400µL RNA Prep buffer to the column. Centrifuge 30sec at 11 000g.
Discard the flow-through and replace the column into the Collection Tube.
SECTION: 5. RNA purification and concentration using RNA Clean and Concentrator-5 kit (ZymoResearch)
25. Transfer the column into a RNA-DNafree microtube (1,5 mL) correctly annotated (sample-template-date).
SECTION: 5. RNA purification and concentration using RNA Clean and Concentrator-5 kit (ZymoResearch)
26. Add 11.5µL DNase/RNase free water directly to the column (and 1 µL of RiboGuard if you store at -80°C). Centrifuge 30sec at 11 000g.
Store at -80°C or proceed directly to cDNA synthesis.