NMR spectroscopy-based metabolomics of organotypic retinal explants

The retina consumes massive amounts of energy, yet its metabolism and substrate exploitation remain poorly understood. Here, we used a murine explant model to manipulate retinal energy metabolism under entirely controlled conditions and utilized 1H-NMR spectroscopy-based metabolomics, in situenzyme detection, and cell viability readouts to uncover the pathways of retinal energy production. Our experimental manipulations resulted in varying degrees of photoreceptor degeneration, while the inner retina and retinal pigment epithelium were essentially unaffected. This selective vulnerability of photoreceptors suggested very specific adaptations in their energy metabolism. Rod photoreceptors were found to rely strongly on oxidative phosphorylation, but only mildly on glycolysis. Conversely, cone photoreceptors were dependent on glycolysis but insensitive to electron transport chain decoupling. Importantly, photoreceptors appeared to uncouple glycolytic and Krebs-cycle metabolism via three di..., Metabolite extraction
After retinal explant culture, at P15, the tissue was quickly transferred into 80% methanol / 20% ethanol, snap-frozen in liquid nitrogen. A sample of the culture medium was taken from the same well plate as the retinal tissue, and snap-frozen in liquid nitrogen. Retinal tissue was placed in 400 µL of methanol (LC-MS grade), transferred to the 2 mL glass Covaris system-compatible tubes and 800 µL of methyl-tert-butyl ether (MTBE) was added, thoroughly mixed, and further subjected to metabolite extraction via ultra-sonication (Covaris E220 Evolution, Woburn, USA). After the extraction, 400 µL of ultrapure water were added for two-phase liquid separation. The aqueous phase was separated and evaporated to dryness. Similarly, 400 µL of the aqueous medium sample was transferred to the 2 mL glass Covaris system-compatible tube, 800 µL of MTBE was added and subjected to the ultra-sonication extraction protocol. Finally, 400 µL of methanol were added and mixed, centrifuge..., , # NMR spectroscopy-based metabolomics of organotypic retinal explants

[https://doi.org/10.5061/dryad.c2fqz61hr](https://doi.org/10.5061/dryad.c2fqz61hr)

The dataset contains raw NMR CPMG spectra files. NMR spectra were recorded at 298 K on a 14.1 Tesla ultra-shielded NMR spectrometer at 600 MHz proton frequency (Avance III HD, Bruker BioSpin, Ettlingen, Germany) equipped with a triple resonance 1.7 mm room temperature micro probe. The spectra were pre-processed using TopSpin 3.6.1 software (Bruker BioSpin).

## Description of the data and file structure

Data contains Carr-Purcell-Meiboom-Gill Sequence (CPMG) experiment spectra files in the pdata 1, .1r folder.

Each folder contains 1 raw NMR spectra file to be opened with Bruker TopSpin software (any current version of the free academic licenced software will be able to open these files).

**The organization of the dataset, the individual data files, and the variables**

The path to follow to open the spectra in each experimental r...