Isolation of Pituitary Cell Nuclei

On the ice, snap-frozen pituitaries were thawed and prepared based on a modified protocol from^{64 (link)}. Same-sex samples were processed simultaneously, with all three female samples processed one day, and all three male samples processed another day. Briefly, RNAse inhibitor (NEB MO314L) was added to the homogenization buffer (0.32 M sucrose, 0.1 mM EDTA, 10 mM Tris-HCl, pH 7.4, 5 mM CaCl2, 3 mM Mg(Ac)2, 0.1% IGEPAL CA-630), 50% OptiPrep (Stock is 60% Media from Sigma; cat# D1556), 35% OptiPrep and 30% OptiPrep right before isolation. Each pituitary was homogenized in a Dounce glass homogenizer (1 ml, VWR cat# 71000-514), and the homogenate filtered through a 40 μm cell strainer. An equal volume of 50% OptiPrep was added, and the gradient centrifuged (SW41 rotor at 17,792 × g; 4 C; 25 min). Nuclei were collected from the interphase, washed, resuspended either in 1X nuclei dilution buffer for snATACseq (10 ×  Genomics) or in 1X PBS/0.04% BSA for snRNAseq, and counted (Invitrogen Countess II).