GSM8464646: Gonadotrope-specific Tgfbr1 conditional knockout male mouse #48B; Mus musculus; RNA-Seq
Pituitary glands were flash frozen in liquid nitrogen and stored at -80C until nuclei isolation Nuclei were isolated from each individual pituitary using OptiPrep Density gradient (STEMCELL Technologies, #07820) and counted using Propidium Iodide staining on a K2 Cellometer (Nexcelome, Lawrence, MA, USA), as described in (27, 60).  Next, single nucleus RNA-seq assay was performed using the Chromium Next GEM Single Cell 3' reagent kit V3.1 (10X Genomics) following manufacturer's instructions. Based on concentrations, nuclei were loaded to target 2,000 to 10,000 nuclei and to generate the single-cell emulsion (Chromium Next GEM Chip G PN-2000177). Reverse-transcription was performed in the emulsion, cDNA amplified, and libraries constructed with v3.1 chemistry. Libraries were indexed for multiplexing (Dual Index Plate TT Set A PN-3000431). Following preparation, libraries were quantified by Qubit 4 fluorometer (Invitrogen) and quality was assessed by Tapestation 4150 (Agilent Technologies, Santa Clara, CA, USA). Equivalent molar concentrations of libraries were pooled. The libraries were then sequenced on a Novaseq X sequencer (Illumina) at Cedars-Sinai Genomics Core using 98+26 paired-end reads.