Chromatin accessibility and microRNA expression in nephron progenitor cells during kidney development [ATAC-Seq]

Mammalian nephron progenitors undergo transcriptional changes over the course of nephrogenesis that sensitize them to signals for differentiation over time. This increases the rate at which they exit their multipotent, self-renewing mesenchymal state and become differentiated epithelial cells, ultimately depleting the progenitor population and leading to the cessation of nephrogenesis. We hypothesized that changes in chromain accessibility and miRNA expression would accompany these transcriptional changes, and that regions of changing chromatin accessibiltiy could reveal chaning regulatory features such as enhancers, including some that affect miRNA expression. To test this, we pooled kidneys from wild-type mouse litters sacrificed at embryonic day 14.5 (E14.5) and post-natal day zero (P0) using positive selection for the surface protein Integrin alpha 8 (Itga8), then sequenced the assay for transposase-accessible chromatin (ATAC-seq) using 50,000 cells, and used the remaining cells for small RNA sequencing (smRNA-seq). ATAC-seq libraries were generated using the Nextera DNA Flex Library Prep Kit (Illumina FC-121-1030) with modifications according to a previously published method (Buenrostro et al., 2013), and all sequencing was performed using an Illumina NextSeq500 by the Health Sciences Sequencing Core at UPMC Children's Hospital of Pittsburgh. Overall design: Sequencing libraries generated using 50,000 nephron progenitor cells isolated and pooled from the same litter of embryos/pups. Isolation performed at E14.5 or P0, and three biological replicates produced per condition (6 samples total).