scRNAseq of RPE-hTert cells expressing different levels of predictive reporter construct

Single cell sequencing of RPE1-hTert cells transduced with the NLS-mVenus-P2A-ER:HRAS^G12V construct, and then flow-sorted into subpopulations based on increasing mVenus intensity, corresponding to different levels of oncogenic RAS stabilised upon induction with 4OHT. RPE1-hTert cells were infected with the predictive reporter (NLS-mVenus-P2A-ER:HRAS^G12V) construct then flow sorted into subpopulations based on increasing mVenus intensity ('S' < 'M' < 'L' < 'XL'). Cells were grown either in monoculture i.e. each subpopulation separately, or in coculture i.e. subpopulations mixed together in equal proportions, and induced with 4OHT. A total of 6 samples (uninduced coculture, induced coculture, induced 'S', induced 'M', induced 'L' and induced 'XL') were hashtagged with TotalSeq-A hashtag antibodies and pooled for encapsulation and single-cell sequencing using the 10x Genomics platform. Induced samples were harvested on Day 6 post-induction.

本数据集针对经NLS-mVenus-P2A-ER:HRAS^G12V构建体转导的RPE1-hTert细胞开展单细胞测序：该细胞经4OHT诱导后，致癌RAS（oncogenic RAS）可发生稳定表达，后续基于mVenus荧光强度的递增梯度，通过流式分选将细胞分为不同亚群，各亚群对应不同水平的稳定致癌RAS表达量。实验中，将RPE1-hTert细胞感染该预测性报告基因NLS-mVenus-P2A-ER:HRAS^G12V构建体后，同样基于mVenus荧光强度递增情况进行流式分选，分为'S'<'M'<'L'<'XL'四个亚群。细胞分别以两种培养方式处理：单培养（即各亚群单独培养）与共培养（即各亚群按等比例混合培养），所有分组均使用4OHT进行诱导。最终共获取6组样本：未诱导共培养组、诱导共培养组、诱导后的'S'、'M'、'L'及'XL'亚群组；所有样本均使用TotalSeq-A标签抗体进行分子标签标记，混合后通过10x Genomics平台完成单细胞包裹与测序。诱导组样本均于诱导后第6天收获。