Wet Lab Protocols

Step-by-step bench protocols written for anyone to use. Each one links to the notebook entries where it was developed and used.

Protocols

Citrate synthase activity (Sigma CS0720, 96-well)
Physiology assays
Astrangia poculata

Measure citrate synthase activity, a proxy for mitochondrial density, in coral host and symbiont fractions as the DTNB reaction rate at 412 nm, normalized to protein.
Coral fixation and decalcification for TEM
Imaging
Galaxea fascicularis

Fix whole coral fragments in zinc formalin, decalcify in HCl, and post-fix single polyps in glutaraldehyde for transmission electron microscopy, keeping the remaining polyps…
Coral tissue removal, homogenization and host/symbiont separation
Tissue & cell prep
Porites astreoides
Astrangia poculata

Airbrush tissue off frozen coral fragments, homogenize the slurry, aliquot it for physiology assays, and split it into host and Symbiodiniaceae fractions.
DNA/RNA co-extraction from coral tissue (Zymo Quick-DNA/RNA Miniprep Plus)
DNA/RNA extraction
Porites astreoides

Extract DNA and RNA from the same small piece of snap-frozen coral (adult fragment or larvae) using bead-beating lysis and Zymo’s paired DNA and RNA spin columns.
ITS2 amplicon PCR for Symbiodiniaceae (incl. touchdown options)
ITS2
Symbiodiniaceae
Porites astreoides

Amplify the Symbiodiniaceae ITS2 region from coral DNA for amplicon sequencing, with high-annealing and touchdown cycling options for low-symbiont (e.g. bleached) samples.
Nanopore direct RNA sequencing on the MinION
Nanopore
Transcriptomics

Workflow notes for native RNA sequencing on an Oxford Nanopore MinION (SQK-RNA002 + R9.4.1 flow cell): input requirements, library prep at a glance, flow cell loading and…
Polar metabolite extraction for LC-MS
Metabolomics
Porites astreoides
Montipora capitata

Extract polar metabolites from snap-frozen coral larvae, juveniles or adult clippings in cold methanol:acetonitrile:water with formic acid, neutralize, and load LC-MS…
S-adenosylmethionine (SAM) ELISA
Physiology assays
Astrangia poculata

Quantify S-adenosylmethionine, the methyl donor for DNA methylation, in coral host tissue by ELISA (BioVision SAM ELISA kit).
Symbiodiniaceae cell density (hemocytometer counts)
Physiology assays
Symbiodiniaceae

Count symbiont cells in coral larvae or tissue homogenates on a hemocytometer, and normalize to larva number or coral surface area.
Total antioxidant capacity (OxiSelect TAC assay)
Physiology assays
Porites astreoides

Measure copper-reducing total antioxidant capacity of coral host tissue against a uric acid standard curve (Cell Biolabs OxiSelect STA-360).
Total protein (alkaline digestion + Pierce BCA assay)
Physiology assays
Porites astreoides

Solubilize protein from coral homogenates or larvae with NaOH, neutralize, and quantify with the Pierce BCA microplate assay.
WGBS library prep (Zymo Pico Methyl-Seq)
DNA methylation
Porites astreoides

Bisulfite-convert 10 ng of coral genomic DNA and build indexed whole-genome bisulfite sequencing libraries with the Zymo Pico Methyl-Seq kit, with QC and a re-amplification…
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Reference protocols

Manuals (PDF)

Kit / reagent File
Zymo Quick-DNA/RNA Miniprep Plus PDF
Zymo EZ DNA Methylation-Gold PDF
Zymo Direct-zol RNA PDF
Qiagen DNeasy Blood & Tissue PDF
Agilent RNA 6000 Pico PDF
Qubit RNA HS PDF
Qubit dsDNA BR PDF
Qubit dsDNA HS PDF
RNAlater PDF
RNAzol RT PDF
RiboPure PDF
Poly(A)Purist PDF
Illumina TruSeq DNA Methylation PDF
Veal et al. 2010 PDF
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