Illumina NovaSeq 6000 paired end sequencing; Single-cell transcriptomics of neonatal mouse anterior pituitary in steady-state conditions and after transgenically inflicted local damage
Single-cell transcriptomics of neonatal 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 Head skin was removed, the cranium opened and the brain withdrawn. The neurointermediate lobe was discarded under a stereomicroscope and the anterior pituitary (AP) collected in medium A. Supernatant was removed and the AP incubated with prewarmed trypsin (2.5%; Gibco) for 15 minutes at 37C. Then, DNase (2 g/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 37C. Supernatant was removed, medium B1 was added, and samples were incubated at 37C. After 5 minutes, medium C was added, and the suspension was again incubated at 37C. 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 g/mL). The resulting suspension was filtered through a 40 m cell strainer; 3% BSA (Serva) was layered at the bottom of the recipient and the sample centrifuged for 10 minutes at 190g. Supernatant was removed, the pellet resuspended in 1 mL of serum-free chemically defined culture medium (SFDM; DMEM/F12 1:1, 5 mg/l human transferrin, 5 mg/l insulin Hybrimax from bovine pancreas, 35 mg/L penicillin, 50 mg/L streptomycin, 600 L/L ethanol, 50 l/L catalase, 1 g/L NaHCO3, 0.5% BSA), and cells were counted using a Coulter Counter Z2 (Beckman Coulter). 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. PD4 GHCre/iDTR mice and Cre-negative control (-/iDTR) 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. The AP was isolated the following day (PD7) and processed as described above. The AP single cell suspensions were converted to barcoded Drop-seq libraries by using the Chromium Single Cell 3 V2 Library, Gel Bead & Multiplex Kit and Chip Kit (10X Genomics; Pleasanton, California, USA), aiming for an estimated 5,000 cells per library. Single cell libraries were prepared with the Chromium Single Cell 3 V2 Chemistry Library Kit, Gel Bead & Multiplex Kit and Chip Kit from 10X Genomics.