title: GSM8262384: control liver, low-grade pancreatic neuroendocrine neoplasm (PaNET), patient #82; Homo sapiens; RNA-Seq
library_construction_protocol: Single cell isolation was performed on a subset of patients with adequate liver tissue as follows: a portion of the specimen was minced with scissors to 1mm pieces and digested in HBSS with 1mg/mL Collagenase IV (Worthington) and 1U/mL DNAse I (Roche) for 20 min at 37˚C with constant agitation. After washing and straining through a 70μm mesh, the non-parenchymal cells (NPCs) were separated by density gradient centrifugation with 40% Optiprep solution as previously described. Specifically, the cell suspension was combined with the Optiprep, underlayed in GBSS buffer, and spun at 900xg for 20 min. The NPCs were concentrated at the interface, were collected and subjected to red blood cell lysis using ACK lysis buffer (Gibco). Isolated NPCs were validated to be >90% CD45+ cells by flow cytometry on BD FACS Canto with the FACSDiva software (BD Biosciences). Analysis was performed using FlowJo v.10 (FlowJo LLC). The isolated hepatic NPC (>90% CD45+) were cryopreserved in 10% DMSO/90% FBS mix until the time of analysis. Upon retrieval, live cells were sorted by exclusion on ethidium homodimer-1 and gating on calcein violet, validated by trypan blue staining, and submitted to downstream processing for scRNAseq if viability was >80%. Library construction was performed according to the manufacturer's instructions (single cell 3' v2 protocol, 10x Genomics)