TITLE: Isolation of clean single-cell samples for physiological or molecular experiments (Radiolaria, Acantharia)
AUTHORS: Joost Mansour, Fabrice Not

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">Many marine protists are not culturable and therefore challenging to study, nonetheless, they are essential in all marine ecosystems. The development of single-cell techniques is allowing for more marine protists to be studied. Thereby Cultivation-independent studies are essential for the study of organisms sensitive to the sampling procedure, such as Radiolaria.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span>This method describes the means to acquire clean isolated single-cells from plankton nets for physiological and/or transcriptomic analysis. The isolation of cells in this protocol is followed by preparing the cells for RNA extraction (protocol: Single-cell total RNA extraction from marine protists (e.g. Acantharia, </span><span style = "font-style:italic;">Strombidium </span><span>cf </span><span style = "font-style:italic;">basimorphum</span><span>, and </span><span style = "font-style:italic;">Prymnesium parvum</span><span>)).</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div></div>

[STEPS]
?. [Preparations]
Prepare filter-sterilised seawater (FSW, 0.2 μm-pore-size)
?. [Preparations]
Prepare your Pasteur pipet isolation tool by elongating the pipet tip under a flame. Carefully break the tip of the pipet to the desired length (and width, longer is thinner).Attach the tubing and optionally a filter on the other end.
?. [Single-cell isolation]
Acquire a sub-sample from a concentrated plankton net sample in a
?. [Single-cell isolation]
Fill another Petri dish with FSW
?. [Single-cell isolation]
Working under aIsolate healthy cells from the sub-sample into the  with clean FSW using a micropipette or modified Pasteur pipette.Dedicate no more than 20 Acantharia cells to a single Petri dish.
?. [Single-cell isolation]
Incubate cells in FSW for .
?. [Single-cell isolation]
Repeat the isolating procedure with a transfer to fresh FSW and  incubation a total of three times.
This procedure allows for self-cleaning of particles attached to the cells and dilution to achieve effective extinction of any contaminating organisms accidentally taken with during isolation.
?. [Single-cell isolation]
These Clean cells can be used for physiological experiments before proceeding to the next step.
?. [Isolation for RNA extraction]
Prepare small plastic zip-locked bags for the samples along with a sample label (pencil on water-proof paper).
?. [Isolation for RNA extraction]
Prepare 100 µL lysis buffer (from RNAqueous kit) in 0.2 mL PCR tubes , label tubes the same as the plastic bags.
on ice
Where possible continue to work on ice
?. [Isolation for RNA extraction]
Transfer one cell per lysis buffer-filled tube and put it in a dedicated small plastic bag (both labeled the same).It is preferred to use a micropipette and 100 µl filter tips. Setting the volume to the minimum allows isolation of the cells with very little residue and a consistent amount of FSW.Immediately freeze in liquid nitrogen.
Disregarding freezing in liquid nitrogen, and proceeding immediately to storage at   has proven to suffice. Though it is still preferred to freeze in liquid nitrogen when possible, especially for physiological experiments.
?. [Isolation for RNA extraction]
Store quickly at
-80 °C
?. [RNA extraction]
Follow the protocol for RNA extraction.Single-cell total RNA extraction from marine protists (e.g. Acantharia, Strombidium cf basimorphum, and Prymnesium parvum).https://www.protocols.io/private/EDB0E27A323211EB952C0A58A9FEAC2A