Transcriptome profiling of Drosophila intestinal cells

Purpose: We isolated Drosophila midgut cells : Delta+ intestinal stem cells (ISCs), Su(H)+enteroblasts (EBs), Esg+ cells (ISC+EB), Myo1A+Enterocytes (ECs), Pros+Enteroendocrine cells (EEs) and How+Visceral muscle cells (VM) from whole midguts to identify stem cell specific genes and study cell type specificities of midgut cells.We also isolated all the cell types from the 5 major regions (R1-R5) of the Drosophila midgut to study differences in cells in different regions.Methods: 3-7 day old female flies were dissected. Flies with GFP/YFP marking different cell types (using the GAL4-UAS system) were used to separate cells of the midgut.The midguts were dissociated with Elastase and FACS sorted using FACS AriaIII. RNA was extracted, amplified and sequenced. Whole midgut samples were sequenced on Illumina GAIIX and regional cell populations were sequenced on HiSeq2000.Methods:Raw fastqc reads were mapped to the Drosophila genome (Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa) using Tophat 2.0.9 at default (using boost_1_54_0, bowtie2-2.1.0, samtools-0.1.19).Methods: For differential expression analysis, DESeq (p-value adjustment 0.05 by method Benjamini-Hochberg) was used. The reads were normalized also to Reads per kilobase of transcript per million mapped reads (RPKM).Results: More than 50% of the genome is expressed in the adult midgut (FlyAtlas- Chintapalli et al., 2007), of these genes about 50% (2457) were differentially expressed (DE) between all 4 cell types (ISCs, EBs, ECs and EEs) atleast 2 folds with 95% confidenceResults: 159 genes that were specifically enriched in ISCs, 509 genes were specifically repressed in ISCsConclusions: Our study represents the first detailed analysis of Drosophila intestinal cell transcriptomes, with biologic replicates, generated by RNA-seq technology.Our data facilitates comparative investigations of expression profiles of cells and reveals novel stem cell genes. Further region specific profiling adds precision to the analysis of variances in the midgut regions. We identify transcriptional regulators and regional transcription factors which modulate the midgut physiology. The dataset will be a great resource for hypothesis generation, tool building and fine tuned studies on the Drosophila midgut.研究目的：我们从完整果蝇中肠中分离出Delta+肠道干细胞（intestinal stem cells, ISCs）、Su(H)+成肠细胞（EBs）、Esg+细胞（ISC+EB）、Myo1A+肠上皮细胞（ECs）、Pros+肠内分泌细胞（EEs）以及How+内脏肌细胞（VM），旨在鉴定干细胞特异性基因并探究中肠细胞的类型特异性特征。同时，我们还从果蝇中肠的5个主要区域（R1-R5）分离了所有上述细胞类型，以研究不同区域细胞间的表达差异。实验方法：选取3-7日龄的雌性果蝇进行解剖。采用通过GAL4-UAS系统标记不同细胞类型的GFP/YFP果蝇，用于分离中肠细胞。使用弹性蛋白酶（Elastase）解离中肠组织，并通过FACS AriaIII进行荧光激活细胞分选。提取总RNA并进行扩增、建库测序：完整中肠样本采用Illumina GAIIX平台完成测序，区域特异性细胞群则采用HiSeq2000平台进行测序。原始fastqc测序reads使用Tophat 2.0.9（默认参数，依赖boost_1_54_0、bowtie2-2.1.0、samtools-0.1.19）比对至果蝇参考基因组（Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa）。差异表达分析采用DESeq工具，通过Benjamini-Hochberg方法将P值校正至0.05。同时，将测序reads标准化为每百万比对reads的每千碱基转录本读数（Reads per kilobase of transcript per million mapped reads, RPKM）。实验结果：成年果蝇中肠中表达的基因占基因组的50%以上（参考FlyAtlas-Chintapalli等，2007），其中约50%的基因（共2457个）在4种细胞类型（ISCs、EBs、ECs和EEs）之间存在至少2倍的差异表达，且置信度达95%。共有159个基因在ISCs中特异性富集，509个基因在ISCs中特异性低表达。研究结论：本研究首次通过RNA测序技术对果蝇肠道细胞转录组进行了详细分析，且设置了生物学重复。本数据集为细胞表达谱的比较研究提供了便利，并揭示了全新的干细胞相关基因。进一步的区域特异性转录组分析提升了中肠区域差异分析的精准度。我们鉴定出了调控果蝇中肠生理功能的转录调节因子及区域特异性转录因子。该数据集将成为果蝇中肠相关假说构建、工具开发及精细化研究的宝贵资源。

搜集汇总

背景与挑战

背景概述

该数据集是一个果蝇（Drosophila melanogaster）中肠细胞的转录组分析数据集，通过RNA-Seq技术对多种细胞类型（如肠道干细胞、肠母细胞、肠细胞等）进行测序，旨在识别干细胞特异性基因和区域特异性表达差异。数据集包含87个样本，总数据量158.47 GB，使用Illumina HiSeq 2000平台生成，公开可访问，为果蝇肠道生物学研究提供了重要资源。