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Hypothalamic cultures at 40 1 days post-differentiation were gently washed twice with calcium and magnesium free DPBS (Thermo Fisher Scientific) before adding dissociation solution consisting of TrypLE Express (Thermo Fisher Scientific) supplemented with papain (Worthington) and 30 M actinomycin D (Sigma) to block transcription as previously described (Chen & Merkle, 2020

The effluent was ionized via ion electrospray in negative mode on a TSQ Quantiva triple-quadrupole mass spectrometry with the following instrument settings: ion spray voltage 2500 V, sheath gas 28, ion transfer tube temperature 325, aux gas 8, and FWHM of 0.7 for both Q1/Q3 resolution