Expression profile of multipotent basal cells and basal-derived luminal cells isolated from the tip region (last 100 µm) of the ductal tree of mouse prostate at P10-P13.

To investigate the molecular reprogramming of epithelial cells of the prostate during basal to luminal differentiation in vivo, we took advantage of the spatial restriction of multipotent basal cells at the distal region (tip - 100 um) of the developing prostate at postnatal day (P)10 (Tika et al., Development, 2019). The distal tips (100 um) of P10-P13 prostate glands were manually dissected from the main ducts under the stereoscope in order to enrich for multipotent basal cells and basal-derived luminal cells. Cell populations were isolated for RNA sequencing based on EpCAM and Cd49f expression via FACS. Prostate tissue of mice at P10-P13 was microdissected under a stereoscope to separate the different lobes. The ventral lobe was used to further separate (by cutting) the tips from the main ducts. Tissues were digested and living cells were isolated using fluorescence-activated cell sorting and analysis. Sorted cells were collected in lysis buffer for RNA extraction. Extracted RNA was sequenced using the bulk RNA-seq strategy.为探究体内前列腺上皮细胞从基底细胞向腔细胞分化过程中的分子重编程机制，我们利用了出生后第10天（P10）发育前列腺远端区域（尖端——100微米处）多能基底细胞的空间限制性分布特征（Tika等，《Development》，2019）。为富集多能基底细胞及基底来源的腔细胞，我们在体视显微镜下，从P10至P13日龄小鼠前列腺腺体的主导管中手动分离远端尖端（100微米区域）。基于上皮细胞黏附分子（EpCAM）与Cd49f的表达特征，通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分离细胞群体以进行RNA测序。我们对P10至P13日龄小鼠的前列腺组织开展体视显微镜下显微解剖，以分离不同前列腺叶；选取腹叶进一步通过切割将尖端与主导管分离。组织经消化后，通过荧光激活细胞分选与分析分离活细胞，将分选得到的细胞收集于裂解缓冲液中用于RNA提取。提取的RNA采用批量RNA测序（bulk RNA-seq）策略完成测序。