title: GSM8806937: TissueCD45+_P1; Homo sapiens; RNA-Seq
library_construction_protocol: Freshly isolated ovarian tissues were processed for scRNA-seq experiments as follows: the tissues were dissociated using enzymatic and mechanical digestion in a gentleMACS Dissociator (Miltenyi, Germany) with RPMI-1640 supplemented with 1%FBS, 2mg/mL collagenase D (Merck, Germany), and 100μg/mL hyaluronidase (Merck). The resulting single-cell suspension was filtered through 100μm and 70μm cell strainers (Corning, USA), washed in HBSS−/− (Euroclone SpA, Italy), and frozen in liquid nitrogen for further analysis. Upon thawing, the cells were stained for live/dead discrimination (Zombie AquaTM Fixable Viability Kit; BioLegend, USA), and with anti-human CD45 monoclonal antibody (mAb) (Clone 5B1; Miltenyi; dilution 1:166). The CD45+ immune cells and tumor-associated CD45- cell counterparts were then sorted by FACS Melody cell sorter (BD Biosciences).PBMCs were isolated using Lympholyte®-H Cell Separation density gradient solution (Cedarlane Laboratories, USA) according to the manufacturer's instructions and were frozen. Upon thawing, PBMC viability exceeded 95% and the cells were immediately processed for scRNA-seq experiments. Libraries for scRNA-seq were prepared following the manufacturer's guidelines using the Chromium Single Cell 3′ Library & Gel Bead Kit v3.1 (10x Genomics). For each sample 10,000 target cells were loaded onto a Chromium single-cell controller (10X Genomics) to generate GEMs. Captured cells were lysed, and the released RNA was barcoded through reverse transcription in individual GEMs. Complementary (c)DNAs were amplified, and their quality was assessed using an Agilent 4200 Tapestation. The scRNA-seq libraries were sequenced with a paired-end 150-bp (PE150) reading strategy, on the Illumina NextSeq500 sequencer for P1 and the Illumina NextSeq2000 sequencer for P2.
identifiers.namespace: GEO
identifiers.id: GSM8806937_r1