Objective:

This Protocol aims to establish a standard Protocol for the sampling of Anemones for the Microbiome Manipulation Experiment , which we will standardize across timepoints.

Broader Context:

The samples taken today (SAM_053-SAM_079) will serve as Timepoint 0 for the initial stability time series experiment on which we will base our manipulations. our aim is to monitor over-all stability, as well as track changes to population based one new lab conditions. is there an influence on microbiome between labs?

Protocol

For Each of the 4 Strains Obtained From Stanford (CC7-endo, CC7-SSB01,CC7-Apo, H2-SSb01) 5 replicates (n=5) were removed from their respective tanks using an individual smart spatula and transfer pipette. Each anemone was pipetted up and down 4-5 times in an unused well of a 24 well plate to remove any algal rings Anemone is then transfered to a pre-filled Bead beating tube with Lysis Matrix A (MP Biomedicals) with minimal water.

500 $\mu$ L of DNA/RNA Shield (Zymo) was added to each tube.

In addtiion to Anemones, .5ml of tank water was sampled from all three Stanford CC7 Strains, and directly from the stock ASW were added to bead Beating tubes with 500 $\mu$ L of DNA/RNA Shield for control purposes.

In future replications we reccomend bead beating at 8.0m/s for 1.5 minutes and centrifuging for about 15 second to ensure full homogenization.

For this Round all samples were bead beaten at 4m/s for 20 seconds, 6.0m/s for two rounds of 20 seconds, and then at 8m/s for 40 seconds. After Bead-beating samples were placed directly into the -80 $^\circ$ C for storage.

A full list of Samples for T0 can be found in the sample spreadsheet in the google drive.