Transcriptome analysis after ectopically expressing KAN1 in SAM epidermis

We ectopically expressed KAN1 in epidermis in ap1 cal background by using AtML1 promoter driven inducible KAN1-2GFP for RNA-Seq studies. Expression of this transgenes in ap1 cal background gives phenotype similar to their expression in wild-type (WT) backgrounds. For pML1>>KAN1-2GFP , we sorted the epidermal cells using FACS, after their induction of 6 hours or 16 hours. As a control we collected the epidermal cells after 6 or 16hr induction of pML1::GR-LHG4 in pML1::BFPer background. Q-PCR analysis on extracted RNA showed the successful sorting and regulation of known target genes of KAN1. Overall design: 2 different transgenic lines (pML1>>REVgto-2Venus, and pML1::GR-LHG4, pML1::BFPer) were used in ap1cal background. 2 time points (6hr and 16hr) were used for each transgenic line. 3-4 biological replicates were used for each time point. cells were collcted through FACS, and used for RNA-Seq analysis

本研究利用AtML1启动子驱动的诱导型KAN1-2GFP载体，在ap1 cal遗传背景的表皮层中异位表达KAN1，以开展RNA-seq相关研究。该转基因构件在ap1 cal遗传背景中的表达表型，与其在野生型（Wild-Type，WT）背景中的表达表型一致。针对pML1>>KAN1-2GFP转基因系，我们在诱导6小时或16小时后，通过荧光激活细胞分选术（Fluorescence-Activated Cell Sorting，FACS）分离表皮细胞。作为对照，我们在pML1::BFPer遗传背景中，对pML1::GR-LHG4诱导6小时或16小时后收集表皮细胞。通过对提取的RNA进行定量PCR（Quantitative PCR，Q-PCR）分析，证实了细胞分选成功，且KAN1的已知靶基因的表达调控效果符合预期。总体实验设计：本实验选用2种不同的转基因系（pML1>>REVgto-2Venus，以及pML1::GR-LHG4与pML1::BFPer），均置于ap1 cal遗传背景中；每个转基因系设置2个时间点（6小时和16小时），每个时间点设置3-4个生物学重复；通过FACS收集细胞后，进行RNA-seq测序分析。