Notch targets in DmD8 and KC Drosophila cells. Drosophila melanogaster

To identify genes upregulated in response to Notch signalling in DmD8 cells. To identify genes upregulated in response to Notch signalling in KC cells. Keywords: Expression analysis at a single timepoint (30' after Notch activation) Overall design: DmD8 cells were obtained from the Drosophila Genomics Resource Center (http://dgrc.cgb.indiana.edu). In the first experiment, 'control cells versus Notch activated cells' mRNA was extracted from DmD8 cells incubated in the absence or presence of EDTA to activate Notch (cells were harvested 30 minutes after addition of EDTA). In a second experiment, 'Notch activated in presenilin inhibited cells versus Notch activated cells', DmD8 cells were split and one 1/2 pretreated with DFK-167 overnight. Untreated and DFK-treated cells were then exposed to EDTA and harvested after 30minutes. RNA was isolated using Trizol (Sigma) and reverse transcription was performed with Superscript III Reverse Transcriptase (Invitrogen) and oligo-dT primers (Sigma). Control and experimental samples were labeled with Cy3 or Cy5, mixed together and hybridized on Drosophila transcriptome long-oligonucleotide microarrays (FlyChip, FL002; http://www.flychip.org.uk/services/core/FL002/). Three biological replicates and dye swaps were performed for each experiment (5 arrays in total/experiment). Slides were scanned by Genepix 400B dual laser scanner (Axon) and spots were found and quantified by Dapple software. KC cells were obtained from Dr Martin Zeidler. Control versus Notch activated KC cells: mRNA was extracted from KC cells incubated in the absence or presence of EDTA to activate Notch (cells were harvested 30 minutes after addition of EDTA). RNA was isolated using TRIzol (Sigma) and reverse transcription was performed with Superscript III Reverse Transcriptase (Invitrogen) and oligo-dT primers (Sigma). Control and experimental samples were labeled with Cy3 or Cy5, mixed together and hybridized on Drosophila transcriptome long-oligonucleotide microarrays (FlyChip, FL002; http://www.flychip.org.uk/services/core/FL002/). Three biological replicates and dye swaps were performed (6 arrays in total/experiment). Slides were scanned by Genepix 400B dual laser scanner (Axon) and spots were found and quantified by Dapple software.

本数据集旨在鉴定DmD8细胞与KC细胞中，响应Notch信号通路（Notch signalling）而上调的基因。关键词：单时间点表达分析（Notch激活后30分钟）。实验整体设计如下：DmD8细胞购自果蝇基因组资源中心（Drosophila Genomics Resource Center，http://dgrc.cgb.indiana.edu）。第一项实验为「对照组细胞 vs Notch激活组细胞」：将DmD8细胞分别于不含EDTA（乙二胺四乙酸，EDTA）与含EDTA的培养基中孵育以激活Notch信号，于添加EDTA 30分钟后收集细胞并提取mRNA。第二项实验为「早老素（presenilin）抑制细胞的Notch激活组 vs 单纯Notch激活组细胞」：将DmD8细胞传代后，取半数用DFK-167预处理过夜。将未处理与经DFK-167处理的细胞均暴露于EDTA中，30分钟后收集细胞。总RNA采用Trizol试剂（Trizol，Sigma）提取，使用Superscript III逆转录酶（Superscript III Reverse Transcriptase，Invitrogen）与寡聚dT引物（oligo-dT primers，Sigma）完成逆转录反应。对照组与实验组样本分别用Cy3、Cy5荧光染料标记后混合，于果蝇转录组长寡核苷酸微阵列（FlyChip FL002；http://www.flychip.org.uk/services/core/FL002/）上进行杂交。每项实验均设置3次生物学重复与染料交换实验，单实验总芯片数为5张。芯片扫描采用Genepix 400B双激光扫描仪（Genepix 400B dual laser scanner，Axon），芯片点信号的识别与定量由Dapple软件完成。KC细胞由Martin Zeidler博士惠赠。KC细胞对照实验（对照组细胞 vs Notch激活组细胞）：将KC细胞分别于不含EDTA与含EDTA的培养基中孵育以激活Notch信号，于添加EDTA 30分钟后收集细胞并提取mRNA。总RNA采用TRIzol试剂（Sigma）提取，使用Superscript III逆转录酶（Invitrogen）与寡聚dT引物（Sigma）完成逆转录反应。对照组与实验组样本分别用Cy3、Cy5荧光染料标记后混合，于果蝇转录组长寡核苷酸微阵列（FlyChip FL002；http://www.flychip.org.uk/services/core/FL002/）上进行杂交。该实验设置3次生物学重复与染料交换实验，单实验总芯片数为6张。芯片扫描采用Genepix 400B双激光扫描仪（Axon），芯片点信号的识别与定量由Dapple软件完成。