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在肾发生过程中会发生转录组变化，随时间推移使其对分化信号更为敏感。这会加快其退出多能、自我更新的间充质状态并分化为上皮细胞的速率，最终耗竭祖细胞群体，导致肾发生过程终止。我们推测，染色质可及性与微小RNA (miRNA) 表达的变化会伴随上述转录组改变，且染色质可及性发生变化的区域可揭示诸如增强子等调控特征的动态变化，其中部分特征会影响微小RNA的表达。为验证该假说，我们收集了野生型同胎小鼠的肾脏：分别在胚胎发育第14.5天（E14.5）与出生后第0天（P0）处死小鼠，通过表面蛋白整合素α8 (Integrin alpha 8, Itga8) 的阳性分选富集肾单位祖细胞，随后取50,000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)。smRNA-seq文库构建采用QIAseq miRNA文库制备试剂盒 (Qiagen 331502)，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平台完成。本次测序文库均提取自同一胎仔/幼崽中分离并混合的肾单位祖细胞，分别在E14.5或P0阶段进行细胞分选，每个实验条件设置3次生物学重复，总计6个样本。