Chromatin accessibility and microRNA expression in nephron progenitor cells during kidney development [miRNA-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). smRNA-seq libraries were generated using the QIAseq miRNA library preparation kit (Qiagen 331502), and all sequencing was performed using an Illumina NextSeq500 by the Health Sciences Sequencing Core at UPMC Children's Hospital of Pittsburgh. Sequencing libraries generated from nephron progenitor cells isolated and pooled from the same litter of embryos/pups. Isolation performed at E14.5 or P0, and three biological replicates were produced per condition (6 samples total)

哺乳动物肾单位祖细胞（nephron progenitors）在肾发生（nephrogenesis）过程中会发生转录组学改变，随时间推移使其对分化信号的敏感性升高。这会加快其退出多能自我更新的间充质状态、分化为上皮细胞的速率，最终耗竭祖细胞群并导致肾发生过程终止。
我们提出假说：染色质可及性与微小RNA（miRNA）的表达变化会伴随上述转录组改变，且染色质可及性发生改变的区域可揭示包括增强子在内的调控特征变化，其中部分调控特征可影响微小RNA的表达。
为验证该假说，我们从胚胎第14.5天（E14.5）和出生后第0天（P0）的野生型小鼠胎仔中采集肾脏，通过表面蛋白整合素α8（Integrin alpha 8, Itga8）的阳性筛选富集肾单位祖细胞并混合；随后取50000个细胞进行转座酶可及性染色质测序（ATAC-seq），剩余细胞则用于小RNA测序（smRNA-seq）。
小RNA测序文库采用QIAseq微小RNA文库制备试剂盒（Qiagen 331502）构建，所有测序工作均由匹兹堡大学医学中心（UPMC）儿童医院健康科学测序中心使用Illumina NextSeq500测序平台完成。
本研究的测序文库均从同一胎胚胎/幼崽中分离并混合的肾单位祖细胞构建；分别在E14.5和P0阶段进行细胞分离，每组设置3次生物学重复，总计6个样本。