GSM7679947: HFD 2; Mus musculus; RNA-Seq
C57BL/6J male mice were placed on control (CTRL) or high fat diet (HFD) for 12 weeks. Pituitary glands were dissected and rinsed in 1x ice cold PBS and were digested using an enzyme cocktail containing 0.25% collagenase in 0.25% trypsin EDTA in HBSS with 25nM HEPES pH 7.2 for 30 minutes at 37C. Enzymatic reaction was stopped with 10% FBS and suspensions were passed through a 70um strainer and centrifuged to pellet cells into single-cell suspensions. Cell suspension was resuspended in 1X PBS in 0.04% BSA and counted. Samples were ran using the 10x Genomics 3' Single Cell Gene Expression assay and protocol. Libraries were prepared according to the manufacturer's instructions (Chromium Next GEM Single Cell 3' v3.1 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscribed to cDNA, which contains an Illumina R1 primer sequence, Feature barcode for cell multiplexing oligo label, Unique Molecular Identifier (UMI) and the 10x Barcode. For the gene expression library construction, the barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction, Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added to ensure that no sample indices overlapped between different libraries.