XIST modulates mammary stem cell homeostasis and tumor fate [RNA-seq]

Disruption of XIST expression in mammary epithelial cells leads to chromatin changes on the inactive X chromosome, transcriptional reactivation of X-linked genes, and defective luminal differentiation. Overall design: Cells from both luminal (ML) and mammary stem cell (MaSC) compartments were isolated from wild-type (WT) and XIST-KO HMLE (Human Mammary Epithelial cells) by FACS sorting based on their positivity for the cell surface antigen ESA (Epithelial Specific Antigen) (ML: ESA+; MaSC: ESA-). Two replicates were generated per compartment and genotype. Total RNA was isolated and sequenced to perform gene expression and allele-specific analyses. Cancer Stem Cells (CSCs) from WT and XIST-KO HMLER were isolated by FACS according to their positivity for CD44 and CD24. Total RNA was isolated and sequenced to perform allele-specific gene expression analyses.

乳腺上皮细胞中X失活特异性转录本（XIST）的表达敲除，可引发失活X染色体的染色质改变、X连锁基因的转录重激活，以及管腔分化异常。

实验设计概述：
从野生型（WT）与XIST敲除（XIST-KO）的人乳腺上皮细胞（HMLE，Human Mammary Epithelial cells）中，通过基于细胞表面抗原上皮特异性抗原（Epithelial Specific Antigen，ESA）的荧光激活细胞分选术（Fluorescence-Activated Cell Sorting，FACS），分离得到管腔细胞群（ML）与乳腺干细胞群（MaSC）：其中ML为ESA阳性（ESA+），MaSC为ESA阴性（ESA-）。每种细胞群与基因型均设置两个生物学重复。提取总RNA并进行测序，以开展基因表达分析及等位基因特异性分析。
从野生型（WT）与XIST敲除的HMLER细胞中，通过基于CD44和CD24阳性表达的荧光激活细胞分选术（FACS），分离得到肿瘤干细胞（Cancer Stem Cells，CSCs）。提取总RNA并进行测序，以开展等位基因特异性基因表达分析。