Single nuclei ATACseq 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 nuclei ATAC library preparation. Nuclei were isolated based on the 10X genomics Nuclei isolation protocol (CG00169 Rev D) with some modifications. We observe that the usage of 0.5ml tubes yields superior nuclei collection. Furthermore, we optimize based on a sample-to-sample basis the time for cell lysis (3-5min). The final lysis buffer concentration for Nonidet P40 was 0.15% over the 0.1% recommendation. Finally, in addition to the final wash with wash buffer, we perform a final wash with the 10X Genomics Nuclei Buffer (PN-2000153/2000207). Nuclei are always kept < 0°C, visually inspected for integrity and quality using a viability dye, prior to library prep which was performed within 30min. Briefly, 5,000-6,500 isolated nuclei were incubated with a transposition mix to preferentially fragment and tag the DNA in open regions of the chromatin. The transposed nuclei were then partitioned into nanoliter-scale Gel Bead-In-emulsions (GEMs) with barcoded gel beads, a master mix, and partition oil on a chromium chip H. Upon GEM formation and PCR, 10x barcoded DNA fragments were generated with an Illumina P5 sequence, a 16nt 10x barcode, and a read 1 sequence. Following library construction, sequencing-ready libraries were generated with addition of P7, a sample index, and a read 2 sequence. Quality controls of these resulting single cell ATAC libraries 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 dual indexing configuration by Illumina NextSeq 2000 (Illumina) to achieve 40,000-30,000 read pairs per nucleus.