GSM7102843: Pituitary_TpitHT; Mus musculus; RNA-Seq
Freshly dissected  pituitaries from Tpit-WT, Tpit-HT and Tpit-KO mice were cut in pieces using a scalpel to facilitate tissue dissociation and kept during the dissections in 1 ml of dissection buffer (DMEM - 10% FBS – 10 mM HEPES, pH 7.4 – 100 U/ml DNase). Pituitaries were then digested in 500 µl of the digestion medium (DMEM – 10 mM HEPES, pH 7.4 - 0.3% BSA – 0.25% trypsin – 100 U/ml DNase) at 37 °C for 10 min. 2 mM EDTA were then added and cells were dissociated by 20-30 ups and downs with a P1000 pipetman homogenizer, following a 5 min incubation at  37 °C. Trypsin was neutralized by adding 10% of FBS (55 µl) and cell dissociation was completed with 10-20 additional ups and downs. Cells were filtered using a 40 µM cell strainer (BF Falcone 352340), centrifuged for 5 min at 400 g and resuspended in 120 µl of FACS buffer (1xPBS - 0.8% BSA – 10% FBS - 10 mM HEPES, pH 7.4 - 100 U/ml DNase) complemented with propidium iodide (1 µg/106 cells) to exclude dead cells and sorted using a FACSAria SORP sorter (BD). Dissociated pituitary cells were processed using Chromium Single Cell 3′ v3 Reagent (10x Genomics) following the manufacturer's instructions. Briefly, 16,000 cells were passed on the channel to recover 10,000 cells. Pituitary cells were partitioned into gel beads in emulsion for cell lysis and barcoded with oligo-dT priming and reverse transcribed. cDNA library was amplified, fragmented and size selected. Samples were controlled at multiple steps during the procedure by running on BioAnalyzer. Resulting libraries were sequenced on Illumina NovaSeq 6000 S1 PE28x91 generating ≈300x106 reads/sample.