TITLE: Collect of Collodarian (Rhizaria, Radiolaria)  nuclei for genomic analyses
AUTHORS: Estelle Bigeard, Loïc Pillet, John Burns, Fabrice Not

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Collodaria are ubiquitous and abundant marine radiolarian (Rhizaria) protists (Biard</span><span style = "font-style:italic;"> et al</span><span>. 2015). They occur as large colonies (a few millimeters up to 3 meters long) or as solitary specimens. Collodarians are known to play an important role in oceanic food webs both as active predators and as hosts of intracellular endosymbiotic microalgae primarily belonging to the dinoflagellate genus </span><span style = "font-style:italic;">Brandtodinium</span><span>. Despite their important ecological roles, very little is known about their diversity and evolution. Taxonomic delineation of collodarians is challenging and only a few species have been genetically characterized.</span></div><div class = "text-block">Most Collodaria form colonies comprising tens to hundreds of individual radiolarian cells (i.e. central capsules) embedded in a gelatinous matrix. Each central capsule contains genomic DNA of the Collodaria host while the gelatinous matrix which also contains the DNA of prey and symbionts.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/74c8ab47edabda576cff2a9e74c9a44e54793fe1ada5d629a38e12e2539cda14/b75mg6iw.png" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/74c8ab47edabda576cff2a9e74c9a44e54793fe1ada5d629a38e12e2539cda14/bm3cg6iw.png" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/74c8ab47edabda576cff2a9e74c9a44e54793fe1ada5d629a38e12e2539cda14/b8qkg6iw.jpg" /></div><div class = "text-block">Some species build a shell-like skeleton aroundtheir central capsule while others have siliceous spicules, similar to those in sponges, in the matrix, and some lack mineral structures altogether. Current taxonomic classification reveals several clades : Sphaerozoidae (skeleton-less but spicule-bearing), Collosphaeridae (mix of skeleton-bearing and skeleton-less taxa), Collophidiidae (skeleton-less). The family Thalassicollidae is composed exclusively of solitary species.</div><div class = "text-block">This protocol describes a method for isolating central capsules containing oly the genomic DNA of the collodarian host by removing prey and symbionts through targeted dissolution of the gelatinous matrix and removal all material outside of host central capsules.</div></div>

[STEPS]
?. [Solutions]
Preparation of Solutions
?. [Solutions]
Sucrose 3M (M = 342.3g / mol)112.96g in 110ml water milliQStore at room temperature
?. [Solutions]
Spermine 0.1M (M = 202.34g / mol)  Powder stored at 4 ° C. Weigh 40.5mg in a 15ml Falcon.then add 2ml water milliQ.Preparation instructions: This product is soluble in water (50 mg / ml), yielding a clear, colorless to light yellow solution. Storage / Stability: Store at 2-8 ° C. Solutions of spermine free base are readily oxidized. Solutions are most stable when prepared in degassed water and stored in frozen aliquots, under argon or nitrogen gas.
?. [Solutions]
Spermidine 0.05M (M = 145.25g / mol)  Liquid stored at room temperature.Weigh 87.15mg in a 15ml Falcon. then add 12ml water milliQ. Preparation instructions: Spermidine is soluble in water (50 mg / ml), ethanol, and ether. Storage / Stability: Spermidine is very hygroscopic and air sensitive. A solution can be formed for storage by dissolving 1.45 g in 10 ml of water and then sterilizing with a 0.22 μm filter. Store this solution as single-use aliquots at -20 ° C for no longer than one month.
?. [Solutions]
4M NaCl  (M = 58.44g / mol)Weigh 0.47g of NaCl. Add 2ml of milliQ water.Store at room temperature.
?. [Solutions]
KCl 5M (M = 74.5513 / mol)Weigh 2.6g of KCl.Add 7ml of milliQ water.Store at room temperature.
?. [Solutions]
EDTA 0.5M pH8  (M = 292.24 / mol)Weigh 15g of EDTA.Add 50ml of milliQ water.Add NaOH pellets until a pH of 8 is reached.QSP 100ml of water milliQ.Filter on 0.2μm.Store at room temperature.
?. [Solutions]
For 500ml of Lysis Buffer Solution    ABCD1  Product
    Initial concentration
    Final concentration
    volume
  2  Sucrose
    3M
    0.3M
    50ml
  3  KCl 
    5M
    60mM
    6ml
  4  NaCl 
    4M
    15mM
    1.875ml
  5  Tris HCl pH8 
    1M
    60mM
    30ml
  6  Spermidine
  
    0.05M
    0.5mM
    5ml
  7  Spermine 
    0.1M
    0.15mM
    750μl
  8  EDTA
    0.5M
    2mM
    2ml
  9  Igepal
  CA-630 
     
     
    2.5ml
  10  Water
  milliQ
     
     
    Qsp 500ml
ABCD1  Product
    Initial concentration
    Final concentration
    volume
  2  Sucrose
    3M
    0.3M
    50ml
  3  KCl 
    5M
    60mM
    6ml
  4  NaCl 
    4M
    15mM
    1.875ml
  5  Tris HCl pH8 
    1M
    60mM
    30ml
  6  Spermidine
  
    0.05M
    0.5mM
    5ml
  7  Spermine 
    0.1M
    0.15mM
    750μl
  8  EDTA
    0.5M
    2mM
    2ml
  9  Igepal
  CA-630 
     
     
    2.5ml
  10  Water
  milliQ
     
     
    Qsp 500ml
?. [Solutions]
For 500ml of Wash Buffer solution    ABCD1  Product
    Initial concentration
    Final concentration
    volume
  2  Sucrose
    3M
    0.3M
    50ml
  3  KCl 
    5M
    60mM
    6ml
  4  NaCl 
    4M
    15mM
    1.875ml
  5  Tris HCl pH8 
    1M
    60mM
    30ml
  6  Spermidine
  
    0.05M
    0.5mM
    5ml
  7  Spermine 
    0.1M
    0.15mM
    750μl
  8  EDTA
    0.5M
    2mM
    2ml
  9  Water
  milliQ
        Qsp 500ml
ABCD1  Product
    Initial concentration
    Final concentration
    volume
  2  Sucrose
    3M
    0.3M
    50ml
  3  KCl 
    5M
    60mM
    6ml
  4  NaCl 
    4M
    15mM
    1.875ml
  5  Tris HCl pH8 
    1M
    60mM
    30ml
  6  Spermidine
  
    0.05M
    0.5mM
    5ml
  7  Spermine 
    0.1M
    0.15mM
    750μl
  8  EDTA
    0.5M
    2mM
    2ml
  9  Water
  milliQ
        Qsp 500ml
?. [Solutions]
Tris HCl 1M pH8  (M = 157.60 / mol)Weigh 15.76g of Tris HCl.Make up to 100ml of milliQ water.Tamp to pH8.Store at room temperature.
?. [Selection of colonies]
Sort several colonies (maximum 20) according to their morphological type (segmented round shape, segmented straight form, non-segmented, purple color, blue dots, etc.) assuming it corresponds to a single species. Observe using a stereomicroscope and take pictures in wide field and zoom.
?. [Lysis of colonies]
In a 6-well plate, deposit 8 ml of Lysis solution per well. Add a 40μm diameter sieve previously rinsed with milliQ water per well. Place about 10 medium sized colonies (1.5 - 2 cm) per sieve. Incubate for 30 minutes at room temperature.  Take a sieve from a well, place it in a 90mm diameter petri dish containing Lysis solution. Tap the sieve and shake circles with the sieve in the BP.Repeat several times until the matrix breaks up and is released from the sieve (about 3 minutes).For purple colonies and those with blue dots, just one lysis will suffice. For other types, perform with a second lysis as below.In a 6-well plate, deposit 8 ml of Lysis solution per well. Place the sieve containing the colonies at the beginning of lysis.Incubate again for 30 minutes at room temperature.
?. [Washing of central capsules]
Remove the sieve from the well and place it in the petri dish containing Wash solution.Tap the sieve and shake circles with the sieve in the BP.Washing of nuclei present in the sieve. Repeat several times until the matrix is completely removed.Observe and control with a binocular loupe (capsule size with respect to the pores, cleanliness of the sample, etc.).Take pictures If OK.
?. [Rinsing & Concentration of nuclei]
Rinse the capsules in 1x PBS solution or in 0.2µm filtered and autoclaved seawater in a 90mm diameter BP.Remove the capsules from the sieve by rinsing it with 0.2µm filtered seawater or 1x PBS solution.Place the capsules in a 1.5ml microtube (or 2ml microtube if necessary).Centrifuge at 1000 rcf 10 minutes.Remove the supernatant.Flash freezer.Storage at -80 ° C.If using of sea water in the above steps, it is better to rinse with PBS before the DNA extraction to remove salt:Rinse with 1x PBS solution by depositing 500 μl of PBS solution into the microtube.Tap gently.Centrifuge at 1000rccf 10 minutes.Remove the supernatant.Repeat once if necessary.
?. [Analyses]
The DNA extraction method will be published under a separate protocols.io (in collaboration with Genoscope).