GSM8676715: CD45+ pituitary cells, newborn; Mus musculus; RNA-Seq
Whole pituitaries or separate posterior and anterior pituitaries were mechanically lysed by suspending and digested with 50 µg/ml DNase 1 (Roche) and 1 mg/ml Collagenase D (Roche) in Hank's Buffered Saline (HBSS; Sigma-Aldrich) at 37 °C for 30-45 minutes. The cell suspension was filtered through silk (pore size 77 µm), pelleted (1006G, 1.5 minutes), and washed with 500 µl of HBSS to remove enzymes. Finally, cells were eluted to Phosphate Buffered Saline (PBS; Sigma-Aldrich) and stained with Fixable Viability Dye eFluor 780 (eBioscience) to label the dead cells. Unspecific binding to low-affinity Fc-receptors was blocked by incubating the cells with unconjugated CD16/32 antibody (BioXCell, clone 2.4G2). Cells were subsequently stained for 30 min at 4 °C with antibodies diluted to the FACS buffer. The cells were sorted with Sony SH800 Cell Sorter (Sony Biotechnology Inc.). Single cells were gated with the FSC-H versus FCS-W plot and the SSC-H versus SSC-W plot to avoid doublets and dead cells were excluded. Live single CD45+ cells were sorted into the RPMI medium containing 2 % FCS. Single cell RNA-sequencing libraries were prepared according to the manufacturer's instructions (CG00052 RevB) using Chromium Single Cell 3′ Library and Gel Bead Kit v2 (10X Genomics, 120237) and Chromium Single Cell A Chip Kit (10X Genomics). Prepared libraries were sequenced using the Illumina Novaseq6000 Sequencing System (RRID: SCR_016387). Library preparation was done in the Single Cell Omics core, and sequencing was done in the Finnish Functional Genomics Center at Turku Bioscience.