GSM4594336: MM_Pit_4-1; Mus musculus; RNA-Seq
For the male samples, the following standard dissociation protocol was used: upon collection, pituitaries were digested with collagenase and dispersed with pancreatin, and the dissociated cells collected acutely. About 700,000-2.5x10^6 cells were collected from single pituitaries and placed in 90% FBS/10% DMSO in individual tubes for cryopreservation and shipped to New York on dry-ice. For the female samples, an improved protocol involving gentle dissociation was used as follows: a) Pituitary removal: Immediately following decapitation, remove the calvarium and gently lift up the cortex from the anterior end, then push it back to reveal the pituitary sitting in the sella turcica. With fine point forceps gently remove the diaphragma sellae covering the pituitary and remove the pituitary. Place it in warm Ca/Mg-free PBS containing 10% c/s FBS. b) Dispersion: Transfer the pituitary to a dish containing Ca/Mg-free PBS (no FBS) and remove the posterior lobe. Rinse 2x in PBS and to a 50 ml centrifuge tube containing a sterile flea-type stir bar and trypsin solution (final concentration at 0.37 mg/ml, Sigma T9935). Gently agitate at 37°C for 10 minutes. After gentle aspiration and expulsion steps, remove the supernatant, add MEM/10%ctFBS and put in an incubator at 37C while continuing the dissociation process. Repeat for 4 cycles, or until no more pieces are visible. Add 5 ml MEM/10%ctFBS and centrifuge. After resuspension, cells are counted and cryopreserved. Upon arrival in New York, all cryopreserved cell samples were stored in liquid nitrogen. On the day of an experiment, the vial was removed from liquid nitrogen on dry-ice, thawed quickly in a 37C waterbath, and cells were serially diluted with 10% FBS /DMEM until a minimum volume of 8ml. Cells were centrifuged at 300g for 5min, resuspended in 1ml of medium and counted. GEM RNAseq was performed as described (10x Genomics, Pleasanton, CA; (24)), following the Single Cell 3' Reagents Kits V2 or V3 User Guides. Cells were filtered, counted on a Countess instrument, and the final concentration was set at 1,000 cells/μl for cells. The amount of cells loaded on the chip was determined based on the 10X Genomics protocol. The 10X chip (Chromium Single Cell 3' Chip kit A v2 PN-12036 or v3 chip kit B PN-2000060) was loaded to target 5,000-10,000 cells final. Reverse-transcription was performed in the emulsion and cDNA was amplified before library construction following the Chromium protocols v2 or v3. Each library was tagged with a different index for multiplexing (Chromium i7 Multiplex kit PN-12062). Quality control and quantification of the amplified cDNA were assessed on a Bioanalyzer (High-Sensitivity DNA Bioanalyzer kit). Sequencing was carried out at the NYGC on Illumina Novaseq using  98+26 paired-end reads.
GEO Accession
GSM4594336