Illumina NovaSeq 6000 paired end sequencing; Single-cell transcriptional profiling of young adult and old mouse anterior pituitary after transgenically inflicted local damage
Single-cell transcriptional profiling of young adult and old mouse anterior pituitary after transgenically inflicted local damage
EXT037_p
After collection, anterior pituitaries were incubated with prewarmed trypsin (2.5%; Gibco) for 15 minutes at 37°C. Then, prewarmed DNase (2µg/mL in medium A; Sigma) was added and flasks were gently moved to mix the content. After APs sank to the bottom (± 1 minute), supernatant was removed and samples were incubated with prewarmed trypsin inhibitor (1mg/mL in medium A; Sigma) for 10 minutes at 37°C. Supernatant was removed, prewarmed medium B1 (see below) was added and samples were incubated at 37°C. After 5 minutes, prewarmed medium C was added and the APs were again incubated at 37°C. The supernatant was removed after 15 minutes and three consecutive rinsing steps with medium C were carried out. APs were then mechanically dissociated using flame-polished Pasteur pipets and transferred to a recipient containing DNase (2µg/mL in medium A). The cell suspension was filtered through a 40µm cell strainer, 3% BSA (Serva) was added to the bottom of the tube and samples were centrifuged for 10 minutes at 190G. Supernatant was removed, the pellet was resuspended in 1mL 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 µL/L ethanol, 50 µl/L catalase, 1 g/L NaHCO3, 0.5% BSA) and cells were counted using the Coulter Counter Z2 (Beckman Coulter). Samples were centrifuged again (10' at 190G) and resuspended in PBS at a concentration of 1.106 cells/mL. Mice were euthanized by CO2 asphyxiation, followed by decapitation. Heads were washed with deionized water and sprayed with 70% ethanol. The skin between the ears was removed, the cranium opened (by breaking the nosebridge and gently removing the skull roof) and brain removed. The neurointermediate lobe was discarded under a stereomicroscope and the anterior pituitary (AP) collected in a 10mL Erlenmeyer flask containing medium A (DMEM, 3 g/L bovine serum albumin (BSA)).  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). 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.