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[DESCRIPTION]
<p>This protocol describes steps and considerations for working in the field to collect samples to be used for molecular work targetting marine (or freshwater) microbial eukaryotes. See downstream protocols for <a href="https://www.protocols.io/view/RNA-and-optional-DNA-extraction-from-environmental-ewnbfde" target="_blank">DNA/RNA extraction</a> and library prep (pending) for 18S rRNA gene tag sequencing.</p>
<p> </p>
<p>Many steps in this protocol can be modified to fit a users' specific needs or environment type. Here, we strive to make recommendations that consider the delicate nature of single-celled eukaryotes and collect samples in the field as cleanly as possible. We use this approach for RNA and DNA-based sequencing - both for tag sequencing and metatranscriptomics.</p>
<p> </p>
<p>This protocol is based off of the field work we conduct at the <a href="https://dornsife.usc.edu/spot/" target="_blank">San Pedro Ocean Time-series (SPOT) station</a>. We go out to the SPOT station once a month. Once we arrive at the SPOT station, we try to sample at the same time of day each month. </p>
<p> </p>
<p>Timelapse video for sampling SPOT: <a href="https://youtu.be/1US3h_qD00w" target="_blank">https://youtu.be/1US3h_qD00w</a></p>
<p> </p>
<p><strong>Materials required</strong>:</p>
<p>(1) 20 L carboys (volume amount flexible)</p>
<p>(2) Covers for carboys (we use pizza bags, black trash bags, or coolers)</p>
<p>(3) Ice packs</p>
<p>(4) Tubing for Niskin </p>
<p>(5) Vacuum pump filtration system (see time-lapse video)</p>
<p>    - trap flask</p>
<p>    - pump</p>
<p>    - extra tubing &amp; rubber/silicon stopper (to fit filter towers)</p>
<p>(6) Filter towers that fit in vacuum pump filtration system</p>
<p>(7) In-line filters, 47 mm</p>
<p>(8) Nitex mesh, cut for 47 mm - 200 um &amp; 80 um</p>
<p>(9) RNase-spray</p>
<p>(10) Absorbent pads (for reducing water spillage &amp; covering countertops)</p>
<p>(11) Liquid nitrogen - make sure to check regulations on transporting liquid nitrogen and storing it on-site/shipboard</p>
<p>(12) 15 mL falcon tubes - RNase/DNase-free</p>
<p>(13) RNA later or Lysis buffer (e.g. RLT buffer from this <a href="https://www.protocols.io/view/RNA-and-optional-DNA-extraction-from-environmental-ewnbfde" target="_blank">protocol</a>)</p>
<p>(14) Filters for collecting material, we use 0.7um GF/F filters*</p>
<p> </p>
<p>*Our reasoning to use this filter size is that the 0.7um nominal pore size will collect the protistan fraction of the community, but it is a woven filter style, so more water can be filtered through without clogging.</p>
<p> </p>
<p> </p>
<p> </p>

[BEFORE_START]
<p><strong>Materials required</strong>:</p>
<p>(1) 20 L carboys (volume amount flexible)</p>
<p>(2) Covers for carboys (we use pizza bags, black trash bags, or coolers)</p>
<p>(3) Ice packs</p>
<p>(4) Tubing for Niskin </p>
<p>(5) Vacuum pump filtration system (see time-lapse video)</p>
<p>    - trap flask</p>
<p>    - pump</p>
<p>    - extra tubing &amp; rubber/silicon stopper (to fit filter towers)</p>
<p>(6) Filter towers that fit in vacuum pump filtration system</p>
<p>(7) In-line filters, 47 mm</p>
<p>(8) Nitex mesh, cut for 47 mm - 200 um &amp; 80 um</p>
<p>(9) RNase-spray</p>
<p>(10) Absorbent pads (for reducing water spillage &amp; covering countertops)</p>
<p>(11) Liquid nitrogen - make sure to check regulations on transporting liquid nitrogen and storing it on-site/shipboard</p>
<p>(12) 15 mL falcon tubes - RNase/DNase-free</p>
<p>(13) RNA later or Lysis buffer (e.g. RLT buffer from this <a href="https://www.protocols.io/view/RNA-and-optional-DNA-extraction-from-environmental-ewnbfde" target="_blank">protocol</a>)</p>
<p>(14) Filters for collecting material, we use 0.7um GF/F filters*</p>

[STEPS]
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