Single cell RNA sequencing of DHT treated human male Islet cells

We analyzed DHT’s effects on the human islet cell transcriptome using single-cell RNA sequencing. We identified the four main endocrine cell subtypes, α, β, δ, pancreatic polypeptide (PPY) cells, as well as ductal, acinar, endothelial, and immune cells by unbiased graph-based clustering. Resulting data was used to evaluate differentially expressed genes across these cell clusters. Human islets (750 IEQ per condition) were treated with either 10nM DHT or EtOH overnight in a humidified incubator containing 4% CO2 at 37°C. Islet cells were then dispersed using TrypLE (Thermofischer), and immediately evaluated for viability (89.88±1.77%) by Cellometer Automated Cell Counter (Nexcelom Bioscience) prior to single cell RNAseq library preparation. For 10x single cell RNAseq library preparation, 5000 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).