Specimens of Pisaster ochraceus were collected in July 2019 from the Bodega Marine Lab intertidal area. They were placed into four treatment groups with five replicates per treatment: a control group, with no dissolved organic matter (DOM) enrichment, and three experimental groups amended with different forms of DOM:
- Peptone (organic nitrogen source usable by most marine bacteria; “Peptone”)
- Cultured phytoplankton (Dunaliela tertiolecta; “Dunaliela”)
- Coastal particulate matter (containing natural phytoplankton, collected from Bodega Bay daily; “Coastal POM”)
All groups were placed on flow-through, large volume sea tables for initial acclimation for seven days. Treatment stars were then exposed to assigned conditions for 15 days. Tube feet were collected at the start of the experiment (day 0), every 48 hours thereafter, and upon termination of the experiment (day 15), and preserved in RNAlater™ (ThermoFisher Scientific; cat. AM7020). Observations were recorded daily on the presence/absence of sea star wasting symptoms, and individuals which succumbed to the disease were subsequently removed from the experiment. We strategically subsampled a collection of tissues from the DOM-enrichment experiment published in Aquino et al. (2021).
RNA was isolated from the tube feet samples using a standard lab TRIzol RNA isolation protocol. Tube feet were removed from RNAlater™ and transferred to new 1.5 ml microcentrifuge tubes, followed by tissue lysis with 1 ml TRIzol reagent (Invitrogen, Carlsbad, CA, USA). To prevent overflow, TRIzol was added in two steps: an initial 500 µl was added to each tube and the tissue was manually homogenized with a plastic pestle, followed by another 500 µl TRIzol, after which samples were vortexed and incubated 5 minutes at room temperature (RT) using a Scientific Industries Vortex Genie 2 and a WVR incubator. Next, to separate the RNA-containing aqueous phase, 200 µl of chloroform was added to the tubes, and the mixture was vortexed for 15 seconds, then incubated for 3 minutes at RT. Following centrifugation at 11.8 rcf for 15 minutes at 4°C, the upper clear, aqueous layer was carefully transferred to a new tube. RNA was precipitated with the addition of 500 µl isopropyl alcohol, then incubated for 10 minutes at RT and centrifuged at 11.8 rcf for 10 minutes at 4°C. After removing the supernatant, pelleted RNA was then washed by adding 1 ml 75% ethanol, followed by a final centrifugation at 7.5 rcf for 5 minutes 4°C. The supernatant was decanted, and the pellet was air-dried for 5 minutes. The RNA product was resuspended in 25 µl H2O and stored at -80°C following quantification with a Qubit® 2.0 Fluorometer and corresponding RNA High Sensitivity (HS) Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). If the concentration exceeded the maximum capacity of the HS kit, quantification was repeated with the Qubit Broad Range (BR) kit.
The isolated RNA was treated with the TURBO DNA-free™ Kit (ThermoFisher Scientific; cat. AM1907) to minimize genomic DNA contamination in our samples using an adapted protocol for routine DNase treatment. First, samples with over 200 ng/µl starting RNA were diluted to meet kit recommendations. Then, 0.1 volume 10X TURBO DNase Buffer and 1 μl TURBO DNase Enzyme were added to the RNA and mixed gently, followed by incubation at 37°C for 30 minutes. Resuspended DNase Inactivation Reagent was then added (2 μl or 0.1 volume, whichever is greater) and the tube was mixed well, then incubated for 5 minutes at RT during which the tube was flicked 2–3 times to redisperse the Inactivation Reagent. Finally, the solution was centrifuged at 10,000 × g for 1.5 minutes, and the supernatant containing the RNA product was transferred to a fresh tube without disturbing the DNase Inactivation Reagent pellet. Samples were again quantified using the same Qubit® procedure and stored at -80°C.
The next step used RNA Clean & Concentrator Kits (Zymo Research, Irvine, CA, USA) to remove inhibitors and other contaminants, including those introduced during the previous step, and to concentrate the RNA product to an ideal volume in preparation for sequencing. Zymo Research offers several different versions of this kit, catered to different ranges of input RNA and desired volume eluent; the Zymo-5 kit (Zymo Research, cat. R1015) has an RNA binding capacity and delivers a more concentrated eluent, while the Zymo-25 kit (Zymo Research, cat. R1018) handles larger amounts of RNA but is limited in its ability to deliver concentrated eluents when input RNA is low. At this stage, 8 samples had undetectable levels of total RNA as measured by Qubit® and the remaining 86 samples had values ranging from 51.51 ng to 13.22 mg. To maximize retention of the isolate in samples with low RNA concentrations while also properly handling samples with high RNA concentrations, the Zymo clean up step into two groups: the first group used the Zymo-5 kit on the 49 samples with less than 600 total ng of RNA and the second used the Zymo-25 kit on the remaining 45 samples with over 600 total ng. Samples were cleaned and concentrated according to the manufacturer’s protocols for either kit (Zymo Research, Irvine, CA, USA).
We quantified RNA one final time in preparation for submission. A total of 12 samples had low total RNA recovered (<250 ng) (from lowest to highest: 13-12, 19-15, 2-0, 17-8, 16-10, 4-8, 18-0, 8-8, 4-4, 8-4, 12-8, 5-12) and 5 had levels of RNA so low they were undetectable (4-0, 13-0, 16-4, 16-8, 16-12). We relayed this information to the sequencing facility but ultimately requested they do their best to amplify sequences regardless. We conducted sequencing through the Genomic Sequencing and Analysis Facility at UT Austin, following submission of 94 samples, we received a set of two read files for each of the 89 successfully sequenced samples (179 read files total).
