Single cell RNAseq profiling of Human Pancreatic Islets across sex

Mechanisms driving sex differences across islet cells is unknown. Thus, studying sex differences in islet regulation and function represent a unique avenue to understand the sex-specific heterogeneity in β cell failure in diabetes. We examined sex and race differences in human pancreatic islets from 15 donors using an orthogonal series of experiments including single cell RNA-seq (scRNA-seq), single nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq), dynamic hormone secretion, and bioenergetics. Human islets (500 IEQ per condition) were cultured overnight in a humidified incubator containing 5% CO2 at 37°C. Islet cells were then dispersed using TrypLE (Thermofischer), and immediately evaluated for viability (90.61±3.04%) by Cellometer Automated Cell Counter (Nexcelom Bioscience) prior to single cell RNAseq library preparation. For 10x single cell RNAseq library preparation, 5000-6500 individual live cells per sample were targeted by using 10x Single Cell 3’ RNAseq technology provided by 10x Genomics (10X Genomics Inc). Briefly, viable single cell suspensions were partitioned into nanoliter-scale Gel Beads-In-EMulsion (GEMs). Full-length barcoded cDNAs were then generated and amplified by PCR to obtain sufficient mass for library construction. Following enzymatic fragmentation, end-repair, A-tailing, and adaptor ligation, single cell 3’ libraries comprising standard Illumina P5 and P7 paired-end constructs were generated. Library quality controls were performed by using Agilent High Sensitive DNA kit with Agilent 2100 Bioanalyzer (Agilent) and quantified by Qubit 2.0 fluorometer (ThermoFisher). Pooled libraries at a final concentration of 750pM were sequenced with paired end single index configuration by Illumina NextSeq 2000 (Illumina).