RNA-seq of in vitro differentiated human abdominal and gluteal adipocyte cell lines following doxycycline induced RSPO3-knockdown

GWAS studies and our own work have identified RSPO3 as a gene modulating human body fat distribution. The GWAS signal at RSPO3 is coincident with an eQTL in mature adipocytes. To assess the effects of RSPO3 on abdominal and gluteal adipocyte biology, we undertook inducible RSPO3-knockdown in in vitro differentiated immortalized human abdominal and gluteal adipocyte cell lines (DFAT cells). Overall design: Abdominal and gluteal DFAT preadipocyte cells stably transduced with the tet-pLKO-puro-shRSPO3 (tet-shRSPO3) or tet-pLKO-puro-shCON (tet-shCON) lentiviral vectors were differentiated in standard adipogenic media. On day 13 of differentiation, cells were treated with doxycycline (final concentration of 0.05 µg ml-1), or vehicle, in hormone-free basal media for ~48 hours. On day 15, cells were harvested for RNA. Total RNA purification and on-column DNase I-treatment were performed using the RNeasy Mini kit (Qiagen). RNA-seq was performed on samples from three independent experiments. In addition to tet-shCON cells, vehicle-treated cells were used as controls.

全基因组关联分析（Genome-Wide Association Study, GWAS）研究与本团队前期工作均已证实RSPO3是调控人体脂肪分布的关键基因。RSPO3位点的GWAS信号与成熟脂肪细胞中的表达数量性状位点（expression Quantitative Trait Locus, eQTL）相吻合。为探究RSPO3对腹部与臀部脂肪细胞生物学特性的影响，本研究在体外诱导分化的永生化人腹部及臀部脂肪细胞系（DFAT细胞）中构建了可诱导RSPO3基因敲低模型。
实验设计方案：将通过慢病毒载体稳定转染tet-pLKO-puro-shRSPO3（tet-shRSPO3）或tet-pLKO-puro-shCON（tet-shCON）的腹部及臀部DFAT前体脂肪细胞，在标准成脂培养基中进行诱导分化。于分化第13天，将细胞置于无激素基础培养基中，用终浓度0.05 μg·mL⁻¹的多西环素或溶剂对照处理约48小时。于分化第15天收集细胞并提取RNA。总RNA纯化与柱上脱氧核糖核酸酶I（DNase I）消化均使用RNeasy迷你试剂盒（Qiagen公司）完成。对来自3次独立重复实验的样本进行RNA测序（RNA-sequencing, RNA-seq）。除tet-shCON细胞外，溶剂对照处理的细胞亦作为对照样本。