Single-cell transcriptomics of elderly mouse anterior pituitary in steady-state conditions and after transgenically inflicted local damage
Single-cell transcriptomics of elderly mouse anterior pituitary in steady-state conditions and after transgenically inflicted local damage
Mice were euthanized by CO2 asphyxiation, followed by decapitation. Heads were washed and sprayed with 70% ethanol. Head skin was removed, the cranium opened and the brain withdrawn. For the -/iDTR and GhCre/iDTR mice, the neurointermediate lobe was discarded under a stereomicroscope. The (anterior) pituitary (AP) was collected in medium A. Supernatant was removed and the AP incubated with prewarmed trypsin (2.5%; Gibco) for 15 minutes at 37°C. Then, DNase (2 µg/mL in medium A; Sigma) was added and the suspension gently mixed. Supernatant was removed and the suspension incubated with trypsin inhibitor (1 mg/mL in medium A; Sigma) for 10 minutes at 37°C. Supernatant was removed, medium B1 was added, and samples were incubated at 37°C. After 5 minutes, medium C was added, and the suspension was again incubated at 37°C. Supernatant was removed after 15 minutes and fragments were washed with medium C. The AP fragments were then mechanically dissociated using a flame-polished Pasteur pipet and transferred to medium A with DNase (2 µg/mL). The resulting suspension was filtered through a 40 µm cell strainer; 3% BSA (Serva) was layered at the bottom of the recipient and the sample centrifuged for 10 minutes at 190 x g. Supernatant was removed, the pellet resuspended in 1 mL of Advanced DMEM/F-12, and cells were counted using a NucleoCounter (Chemometec). The cell suspension was centrifuged again (10 minutes at 190g) and resuspended in PBS with 0.04% BSA at a concentration of 1x10^6 cells/mL.  GHCre/iDTR mice and Cre-negative control littermates were intraperitoneally injected with 4 ng DT/g bodyweight (Sigma-Aldrich, Bornem, Belgium) twice a day (8 h in between the injections) for 3 consecutive days. Pituitaries were isolated and analyzed the following day (day 4). The AP single-cell suspensions were converted to barcoded Drop-seq libraries by using the Chromium Single Cell 3' V3.1 Library, Gel Bead & Multiplex Kit and Chip Kit (10X Genomics; Pleasanton, California, USA), aiming for an estimated 8,000 cells per library. Single-cell libraries were prepared with the Chromium Single Cell 3' V3.1 Chemistry Library Kit, Gel Bead & Multiplex Kit and Chip Kit from 10X Genomics.