TITLE: Plant-associated microbiome sampling protocol for field work
AUTHORS: Cassie Ettinger, Marina LaForgia

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details sampling of plant-associated microbiomes from plant communities in field plots.</div></div>

[STEPS]
?. [Microbiome Sampling Protocol]
Take a photo of the subplot plot or subplot to be sampled
a. Make sure the photo is GPS tagged!.   b. From the photo, we can later get time of sampling and approximate GPS location.
?. [Microbiome Sampling Protocol]
Take any environmental measurements at this time (e.g. temperature, soil moisture).
?. [Microbiome Sampling Protocol]
Put on new gloves.
?. [Sampling Plots]
Using a bulb planter, core the subplot.
a.Try to get a representative sample that includes all species of interest.b. If plant community composition is important, make sure to note which species were included in the core and which were missing
?. [Sampling Plots]
Push core out into new labeled ziplock bag using a sterile scoopula or gloved hands (replace gloves after sampling if hands are used)
?. [Processing Samples]
Take plant species out of bag and shake bulk soil off of roots, what soil remains on the plant constitutes as the rhizosphere soil
If individual plant species is important to research questions and identification and separation of individuals is possible, then separate individual plant species and sample each species individually for next steps.
?. [Sampling Plots]
Spray the inside of the bulb planter with EtOH and wipe down with paper towels (or alcohol wipes) between uses.
?. [Sampling Plots]
Can stop here and place bags  for transport back to lab for processing or can immediately proceed and process in field.
on ice
?. [Root Sample]
Using sterilized scissors, cut roots off of aboveground biomass and place in a labelled  tube.
1.5 mL
If tons of roots or large roots, only use 1-3 from each species in core.
?. [Leaf Sample]
Using sterilized scissors, cut leaves off of specimens using sterilized scissors and place in labelled  tube.
1.5 mL
a. Standardize sampling of leaf age and position between plants depending on research question.b. Only need ~1-3 cm section of leaf tissue.
?. [Bulk Soil Sample]
Shake remaining soil in bag to homogenize.
?. [Bulk Soil Sample]
Using sterilized scoopula, put soil in a labelled  tube.
1.5 mL
?. [Transport and clean up]
Place all samples ( conical, tube of roots, tube of leaves, tube of soil, bag of soil)  for transport or directly into  or  freezer in processing in lab.
50 mL
on ice
-20 °C
-80 °C
?. [Example Note Pages]
Example note pages:  Plot    Suplot    Photo    Species Cored    Collector    Date    Notes    92  a    X         Cassie         Gopher destroyed plot  b    X    Marina       c  X                 d  X                 e  X                 control  X    none
?. [Bulk Soil Sample]
Fill soil as much as possible, but at least halfway.
?. [Rhizosphere Sample]
Swirl and shake roots in  conical (prefilled with autoclaved sterile water) to collect soil associated with roots.
50 mL
?. [Rhizosphere Sample]
Swirl/shake for ~.
Note: post processing of this sample to get a rhizosphere pellet will be needed later in lab. This involves centrifuging    conical tubes (e.g. at 4000 g  for  ), decanting excess water and then using a flame sterilized scoopula to take ~250 mg of the soil pellet for DNA extraction. Alternatively - one can take subsample(s) from the    conical tubes by first shaking/vortexing tubes and then transferring   into    eppendorf tube to use with a benchtop centrifuge (e.g. at 10,000 g for    ). This procedure is modified from Edwards et al (2015).
?. [Transport and clean up]
and repeat for all plots sampling
?. [Transport and clean up]
Replace gloves and make sure to clean bulb planter/scoopulas with EtOH.