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URL: https://dx.doi.org/DOI:10.17504/protocols.io.hisb4ee

Authors: Sarah Hu

Group: Protist Research to Optimize Tools in Genetics (PROT-G), Caron Lab - Protistan Ecology, SCOPE

Summary: 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 DNA/RNA extraction and library prep (pending) for 18S rRNA gene tag sequencing.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.This protocol is based off of the field work we conduct at the San Pedro Ocean Time-series (SPOT) station. 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. Timelapse video for sampling SPOT: https://youtu.be/1US3h_qD00wMaterials required:(1) 20 L carboys (volume amount flexible)(2) Covers for carboys (we use pizza bags, black trash bags, or coolers)(3) Ice packs(4) Tubing for Niskin (5) Vacuum pump filtration system (see time-lapse video)    - trap flask    - pump    - extra tubing & rubber/silicon stopper (to fit filter towers)(6) Filter towers that fit in vacuum pump filtration system(7) In-line filters, 47 mm(8) Nitex mesh, cut for 47 mm - 200 um & 80 um(9) RNase-spray(10) Absorbent pads (for reducing water spillage & covering countertops)(11) Liquid nitrogen - make sure to check regulations on transporting liquid nitrogen and storing it on-site/shipboard(12) 15 mL falcon tubes - RNase/DNase-free(13) RNA later or Lysis buffer (e.g. RLT buffer from this protocol)(14) Filters for collecting material, we use 0.7um GF/F filters**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.

Affiliations: University of Southern California

Version: 1

Publication Date: 2017

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