scRNAseq of Aging Drosophila ISCs [NGS1910]

Tissue homeostasis requires long-term lineage fidelity of somatic stem cells. Whether and how age-related changes in somatic stem cells impact the faithful execution of lineage decisions remains largely unknown. Here, we address this question using genome-wide chromatin accessibility and transcriptome analysis as well as single cell RNA-seq to explore stem cell intrinsic changes in the aging Drosophila intestine. These studies indicate that in stem cells of old flies, promoters of Polycomb (Pc) target genes become differentially accessible, resulting in the increased expression of enteroendocrine (EE) cell specification genes. Consistently, we find age related changes in the composition of the EE progenitor cell population in aging intestines, as well as a significant increase in the proportion of EE-specified ISCs and progenitors in aging flies. We further confirm that Pc-mediated chromatin regulation is a critical determinant of EE cell specification in the Drosophila intestine. Pc is required to maintain expression of stem cell genes while ensuring repression of differentiation and specification genes. Our results identify Pc group proteins as central regulators of lineage identity in the intestinal epithelium and highlight the impact of age-related decline in chromatin regulation on tissue homeostasis. For the scRNA-seq experiments, we used the wild-type WDah fly line and dissected whole midguts at young (~7d), mid-age (~30d) and old time-points (~60d). In total, we had two young, one mid age and two old samples. The cell density and viability of single-cell suspension were determined by Vi-CELL XR cell counter (Beckman Coulter). All of the processed samples had very high percentage of viable cells (>90%). The cell density was used to impute the volume of single cell suspension needed in the reverse transcription (RT) master mix, aiming to achieve ~6,000 cells per sample. cDNAs and libraries were prepared following manufacturer’s user guide (10x Genomics). Libraries were profiled on 2100 Bioanalyzer (Agilent Technologies) using High Sensitivity DNA kit (Agilent Technologies) and quantified using Kapa Library Quantification Kit (Kapa Biosystems). Each library was sequenced in one lane of HiSeq4000 (Illumina) following manufacturer’s sequencing specification (10x Genomics).