GSM9853720: Pituitary cells; Homo sapiens; RNA-Seq
Nuclei were isolated from a CCS pituitary tumor sample previously stored in liquid nitrogen using a mortar-based homogenization method, followed by purification via iodixanol (Sigma, D1556-250ML) density gradient centrifugation. Quality control included nuclei counting with AOPI staining on a Countstar S2 instrument and assessment of nuclei integrity by trypan blue staining under a microscope. Library construction was performed using the Chromium Next GEM Single Cell 3' Kit v3.1 (10X Genomics) on the Chromium Controller platform. Approximately 25,410 nuclei (30 µL at 847 nuclei/µL) were loaded, targeting 10,000 nuclei capture. Gel Beads-in-emulsion were generated with near-saturation loading of barcoded beads, enabling hybridization of polyadenylated RNA to the beads upon lysis. Reverse transcription was carried out to synthesize cDNA tagged with cell barcodes and unique molecular identifiers (UMIs), followed by second-strand synthesis, adapter ligation, and universal amplification according to the manufacturer's protocol. Libraries were quantified using an Agilent 2100 Bioanalyzer with a High Sensitivity DNA Chip and a Qubit High Sensitivity DNA Assay kit (Thermo). Sequencing was performed on an Illumina NovaSeq X Plus platform in PE150 mode, targeting 20,000 reads per nucleus and a total output of 100 Gb.