TITLE: Single-cell total RNA extraction from marine protists (e.g. Acantharia, Strombidium cf basimorphum, and Prymnesium parvum)
AUTHORS: Joost   Mansour, Konstantinos Anestis, Fabrice Not, Uwe John

[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. Such genomic approaches aim to help to disentangle heterotrophic processes such as phagotrophy from osmotrophy and phototrophic-induced anabolic activities. This information will then support cellular and metabolic modeling by better elucidating the physiological mechanisms and quantifying their importance in different scenarios.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">However, single-cell protocols and low input RNA kits for transcriptomics are usually made for and tested with mammalian cells, as such the feasibility and efficiency of single-cell transcriptomics on highly diverse mixotrophic protists are not always known. Often single-cell transcriptomics of microbial eukaryotes shows low transcript recovery rates and large variability.</div></div><div class = "text-block"><span>We report on transcriptomic methods that we have successfully performed on single cells of 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></div><div class = "text-block"><span>This first protocol outlines the total RNA extraction step utilizing the</span><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Reagent and general preparations]
Samples need to have been acquired in  (from the ). See our sampling protocol here:dx.doi.org/10.17504/protocols.io.bqvrmw56
100 µl
?. [Reagent and general preparations]
Prepare and label two RNase-free tubes per sample, one tube for the total RNA extract and one tube for an aliquot for the Bioanalyzer.
?. [Reagent and general preparations]
Prepare   of Elution Solution (from ) in  a and put the aliquot in a  heat block.
[times number of samples]
75 °C
?. [Sample preparations]
For Acantharia samples: thaw and vortex 2 times  all samples (be sure to secure the lids).
For other samples types just thaw   , and continue with step 6.
?. [Sample preparations]
Spin down gently
?. [Sample preparations]
for
75 Room temperature
?. [RNA extraction]
Add  to all samples
50 µl
?. [RNA extraction]
Brief vortex and gentle/short spin down
?. [RNA extraction]
Load the () samples each onto a separate Microfilter cartridge (use 100 µL pipet twice)
150 µl
?. [RNA extraction]
Centrifuge    (i.e. at maximum)
Centrifuge: 10000 34, 10 s
?. [RNA extraction]
Add of Wash Solution 1 unto the column (Microfilter cartridge)
180 µl
?. [RNA extraction]
Centrifuge    (i.e. at maximum)
Centrifuge: 10000 34, 30 s
?. [RNA extraction]
Add   of Wash Solution 2/3 unto the column (Microfilter cartridge)
180 µl
?. [RNA extraction]
Centrifuge    (i.e. at maximum)
Centrifuge: 10000 34, 30 s
?. [RNA extraction]
Repeat addition of  of Wash Solution 2/3 unto the column (Microfilter cartridge)
180 µl
?. [RNA extraction]
Centrifuge    (i.e. at max)
Centrifuge: 10000 34, 30 s
?. [RNA extraction]
Remove and discard all flow-through (pour it out) from the collection/elution tube
?. [RNA extraction]
Centrifuge     (i.e. at maximum) to remove any liquid still on the filter
Centrifuge: 10000 34, 1 min
?. [RNA extraction]
Replace the collection/elution tube with a clean new one (keeping the top part with filter and RNA!)
?. [RNA extraction]
Add Elution Solution (pre-heated at )
6.5 µl
75 °C
Do this directly to the center of the filter
?. [RNA extraction]
Incubate at  for
75 Room temperature
?. [RNA extraction]
Centrifuge    (i.e. at max)
Centrifuge: 10000 34, 30 s
?. [RNA extraction]
Add Elution Solution (pre-heated at )
6 µl
75 °C
Do this directly to the center of the filter
?. [RNA extraction]
Incubate at  for
75 Room temperature
?. [RNA extraction]
Centrifuge    (i.e. at max)
Centrifuge: 10000 34, 30 s
?. [RNA extraction]
Transfer your  RNA from the Elution tube to a (labeled)  (use 100 µL pipet).
12.5 µl
?. [RNA extraction]
For Bioanalyser take an aliquot of 1.5 µL in a separate (labeled) tube, still. Analyze immediately or store.
on ice
?. [RNA extraction]
Store RNA at
-80 °C
?. [Sample verification]
Attempt to quantify and assess the quality of total RNA extract using Bioanalyzer.Follow the manufacture's protocol for the Agilent RNA 6000 Pico Kit.
Using small single-celled protists RNA is often not measurable.
?. [Sample verification]
Total RNA extracted from single cells are often low in concentration and yield. And could in most cases not be measured (e.g. for Strombidium and Prymnesium).For Acantharia (±80 µm diameter) its was measurable but could still be, as low as With these single-cell low RNA concentrations, the total RNA extract would anyway be used in subsequent protocols without dilution making accurate quantification of concentrations incidental, and this also minimized cost and workload.
?. [Sample verification]
?. [Sample verification]
?. [Subsequent protocol:]
Follow up with cDNA synthesis and library preparation for Illumina sequencing
{"blocks":[{"key":"apkl","text":"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. Such genomic approaches aim to help to disentangle heterotrophic processes such as phagotrophy from osmotrophy and phototrophic-induced anabolic activities. This information will then support cellular and metabolic modeling by better elucidating the physiological mechanisms and quantifying their importance in different scenarios.","type":"align-left","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"b4oae","text":"However, single-cell protocols and low input RNA kits for transcriptomics are usually made for and tested with mammalian cells, as such the feasibility and efficiency of single-cell transcriptomics on highly diverse mixotrophic protists is not always known. Often single-cell transcriptomics of microbial eukaryotes shows low transcript recovery rates and large variability.","type":"align-left","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"ck39t","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"cvfm9","text":"We report on transcriptomic methods that we have successfully performed on single cells of Acantharia, Strombidium basimorphum, and Prymnesium parvum. ","type":"unstyled","depth":0,"inlineStyleRanges":[{"length":25,"offset":103,"style":"italic"},{"length":19,"offset":132,"style":"italic"}],"entityRanges":[],"data":[]},{"key":"efflf","text":"This protocol follows up after total RNA extraction (from the protocol at dx.doi.org\/10.17504\/protocols.io.bp6xmrfn) to prepare cDNA libraries for Illumina sequencing. The described protocol uses the SMART-Seq4 kit (Takara #634891) for cDNA synthesis and amplification, but this can also be successfully performed with the NEBNext kit (NEB #E6421). The NEBNext kit protocol is very similar to the protocol described here and generally the manufacture's protocol can be followed but see the notes at step 4 and step 18 of this protocol, and do the final elution after cDNA purification in 10 mM Tris (pH 8.0).","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[{"key":0,"length":41,"offset":74}],"data":[]},{"key":"cgunc","text":"The subsequent cDNA library is prepared following the  . ","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[{"key":1,"length":1,"offset":54}],"data":[]},{"key":"75ia3","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"f146t","text":"  ","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[{"key":2,"length":1,"offset":0}],"data":[]},{"key":"cogtp","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[{"type":"link","data":{"guid":"BF008CFB621B11EB82BF0A58A9FEAC2A","url":"https:\/\/dx.doi.org\/10.17504\/protocols.io.bp6xmrfn"}},{"type":"reagents","data":{"can_edit":0,"cas_number":"","guid":"69F1065032F211EBA38297F0DB0378D3","id":124708,"in_steps":null,"linfor":"","mol_weight":0,"name":"Nextera XT DNA Library Preparation Kit","public":0,"rrid":"","sku":"FC-131-1096","url":"https:\/\/www.illumina.com\/products\/by-type\/sequencing-kits\/library-prep-kits\/nextera-xt-dna.html","vendor":{"name":"illumina","affiliation":null,"affiliations":[],"username":null,"note":null,"link":"http:\/\/www.illumina.com","image":{"source":"https:\/\/www.protocols.io\/img\/vendors\/placeholder.png","placeholder":"https:\/\/www.protocols.io\/img\/vendors\/placeholder.png"},"badges":[],"verified":0,"is_verified_user":false,"research_interests":null,"blocked_by_you":false,"blocked_you":false,"hide_following":false,"id":245},"stats":{"total_protocols":0}}},{"type":"image","data":{"guid":"B87C3450332B11EBA38297F0DB0378D3","height":472,"id":119323,"is_video":false,"legend":"","mime":"image\/png","original_name":"g3662.png","placeholder":"https:\/\/s3.amazonaws.com\/protocols-files\/public\/674809e005421a3be89d2f4eb645e007308aad04344d512dc0aa0dc6b5caa402\/csc836c6.png","source":"https:\/\/s3.amazonaws.com\/protocols-files\/files\/dws536c6.jpg","type":1,"webp_source":"https:\/\/s3.amazonaws.com\/protocols-files\/files\/dws436c6.webp","width":550}}]}