J Clin Invest.
Study design for determination of the transcriptome and differential histone marks in sorted human islet cells.
(A) Human islets were dispersed and subjected to FACS to obtain cell populations highly enriched for α, β, and exocrine (duct and acinar) cells. Chromatin was prepared and precipitated with antibodies for H3K4me3 and H3K27me3 followed by high-throughput sequencing (ChIP-Seq) (H3K4me3: n = 4 α, n = 4 β, n = 2 exocrine, H3K27me3: n = 3 α, n = 3 β, n = 2 exocrine). RNA-Seq analysis was performed to determine mRNA and lncRNA levels (n = 3 α, n = 3 β, n = 2 exocrine). (B) Sample purity assessment. Normalized insulin and glucagon expression levels of the individual α and β cell populations were obtained by qRT-PCR to calculate the contamination by the opposite cell population, revealing high sample purity (2.5%–10.3% contamination in the α and 2-13.1% contamination in the β cell populations; details in Supplemental Methods). (C) Analysis pipeline for H3K4me3 and H3K27me3 ChIP-Seq data. Peak calling (H3K4me3: GLITR; H3K27me3: STAR) on individual replicates, followed by signal pooling, was employed to assess histone modification profiles of α, β, and exocrine cells. Heat map analysis confirmed reproducibility of replicates. (D) Genome browser image of the PDX1 locus showing H3K4me3 enrichment in α, β, and exocrine cells and H3K27me3 enrichment only in α cells (defined as monovalent H3K4me3 enrichment in β and exocrine cells, bivalent mark in α cells; CpG islands: red bars).