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[DESCRIPTION]
<p>This protocol describes the extraction process of RNA and/or DNA from marine environmental samples. Samples are assumed to be collected via filtering sample seawater onto filters. Filters are then flash frozen in the first lysis buffer. The protocol describes the steps used to lyse and separate material from filters and use the lysate for RNA and/or DNA extraction using Qiagen extraction kits (e.g. https://www.qiagen.com/us/shop/Sample-Technologies/Combined-Sample-Technologies/).</p>
<p> </p>
<p>Using this protocol, RNA and DNA has been used for molecular analyses such as tag sequencing (16S or 18S), (meta)genomic, or (meta)transcriptomic applications</p>
<p> </p>
<p>See other Caron lab protocols that <a href="https://www.protocols.io/view/sample-collection-from-the-field-for-downstream-mo-hisb4ee" target="_blank">detail field work sampling </a>and downstream PCR amplification/libray prep for microbial eukaryotic-specific tag sequencing (18S rRNA gene).</p>
<p><br />List of materials needed before starting is in <strong>Guidelines.</strong></p>

[BEFORE_START]
<ul>
<li>Please read through all of the warnings, steps, and considerations for Qiagen extraction kit</li>
<li>Ensure that Buffer RLT+ (or RLT) has beta mercaptoethanol added, as per the Qiagen instructions</li>
<li>Consider filter type (or however you are collecting cellular material) and compatibility to reagents (RLT+ buffer)</li>
</ul>

[GUIDELINES]
<p>These steps for RNA (&amp; DNA) extraction are specific to samples of seawater filtered onto GF/F, nitex mesh, or polycarbonate filters*. This protocol focuses on additional steps taken for lysis and collection of material from filters before using a Qiagen DNA/RNA Allprep kit.</p>
<p> </p>
<p>See other Caron lab protocols that <a href="https://www.protocols.io/view/sample-collection-from-the-field-for-downstream-mo-hisb4ee" target="_blank">detail field work sampling</a> and downstream PCR amplification/libray prep for microbial eukaryotic-specific tag sequencing (18S rRNA gene).</p>
<p> </p>
<p>Considerations:</p>
<ul>
<li>*Please confirm/test that protocol chemistry is compatible with filter of your choice. The beginning of this protocol assumes that collected filters were flash frozen into RLT+ Buffer (Qiagen)</li>
<li>This protocol is designed to also be compatible with other Qiagen extraction protocols. Please read all steps of the protocol ahead of time to confirm other Qiagen components are interchangeable.
<ul>
<li>Ensure loading material is within the range of extraction kit (see Qiagen manual)</li>
</ul>
</li>
<li>When working with RNA please minimize any exposure to non-RNase DNase safe environments and prior to lysing, ensure samples remain frozen at -80C.</li>
</ul>
<p> </p>
<p><strong>Materials</strong>:</p>
<ul>
<li>Qiagen extraction kit, can be from a variety of kits</li>
<li>RNase-free DNase set from Qiagen (cat # 79254)</li>
<li>Ice</li>
<li>Heat block (65C)</li>
<li>RNase-DNase clean pipets and filter pipet tips</li>
<li>gloves and RNase/DNase-free bench space</li>
<li>5mL sterile syringe, 1 needed for each filter* (if using filter extraction)</li>
<li>5mL centrifuge tubes, RNase-DNase free (additional UV-sterilization preferred)</li>
<li>0.5 mm Silica beads (e.g. Biospec products), made RNase-DNase free. Beads can be baked in oven to make RNase/DNase-free for 2 hrs at 550F.</li>
<li>vortex</li>
<li>Microcentrifuge, both desktop mini and larger centrifuge to fit 1.5mL tubes</li>
<li>96-100% EtOH</li>
<li>70% EtOH (Freshly made in sterile water)</li>
<li>Sterile forceps (or disposable forceps)</li>
</ul>

[STEPS]
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