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Gene Expression Analysis of Sorted Cells by RNA-seq in Drosophila Intestine
采用RNA-seq法分析果蝇肠道中分选细胞的基因表达   

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Abstract

RNA sequencing (RNA-seq) has become a popular method for profiling gene expression. Among many applications, one common purpose is to identify differentially expressed genes and pathways in different biological or pathological conditions. This protocol provides detailed procedure for RNA-seq analysis of ~250,000 sorted Drosophila intestinal cells (Chen et al., 2016), in which RNA amplification is not required.

Keywords: RNA-seq(RNA-seq), FACS(FACS), Drosophila intestine(果蝇肠道), Progenitor cells(祖细胞), Transcriptome analysis(转录组分析)

Background

Transcriptome analysis by RNA-seq has become a popular method for the identification of differentially expressed genes and pathways under different biological or pathological conditions. For samples that yield low mRNA levels, RNA or cDNA amplification was commonly performed before deep-sequencing (Dutta et al., 2015). However, this procedure could potentially omit important candidates that are expressed in low abundance. Here we provide a detailed procedure for RNA-seq analysis of sorted Drosophila gut cells in which RNA amplification is not required.

Materials and Reagents

  1. Isolation of intestinal progenitor cells by FACS
    1. Microcentrifuge tube (Corning, Axygen®, catalog number: MCT-150-C )
    2. 40 μm filters
    3. 70 μm filters (Corning, Falcon®, catalog number: 352350 )
    4. The fly strains carrying Esg-GFP fluorescent marker
    5. Elastase (Sigma-Aldrich, catalog number: E0258 )
    6. RNAiso Plus (Takara Bio, catalog number: 9108/9109 )
    7. NaCl
    8. KCI
    9. Na2HPO4
    10. KH2PO4
    11. Diethyl pyrocarbonate (DEPC) (Sigma-Aldrich, catalog number: D5758 )
    12. 1x DEPC-PBS (see Recipe)
    13. Elastase solution (see Recipe)

  2. RNA isolation
    1. Directzol RNA MiniPrep Kit (ZYMO RESEARCH, catalog number: R2050 )
    2. 95-100% ethanol

  3. Library construction
    1. Agilent RNA 6000 Pico Kit (Agilent Technologies, catalog number: 5067-1513 )
    2. Dynabeads® mRNA DIRECTTM Purification Kit (Thermo Fisher Scientific, AmbionTM, catalog number: 61011 )
    3. Hexadeoxyribonucleotide mixture, pd(N)6 (Takara Bio, catalog number: 3801 )
    4. dNTP mixture (Takara Bio, catalog number: 4019 )
    5. DTT (provided by SuperScript® II reverse transcriptase)
    6. Recombinant RNasin® ribonuclease inhibitor (Promega, catalog number: N2511 )
    7. SuperScript® II reverse transcriptase (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18064014 )
    8. Second-strand buffer (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10812014 )
    9. DNA polymerase I (Takara Bio, catalog number: 2130A )
    10. RNase H (New England Biolabs, catalog number: M0297S )
    11. Agencourt AMPure XP Kit (5 ml) (Beckman Coulter, catalog number: A63880 )
    12. NEBNext® DNA library prep master mix set for Illumina® (New England Biolabs, catalog number: E6040L )
    13. NEBNext® multiplex oligos for Illumina® (New England Biolabs, catalog number: E7335S / E7500S )
    14. Agilent High Sensitivity DNA Kit (Agilent Technologies, catalog number: 5067-4626 )
    15. Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32851 )
    16. Illumina Library Quantification Kit (Kapa Biosystems, catalog number: KK4824 )

Equipment

  1. Isolation of intestinal progenitor cells by FACS
    1. Dissecting microscope with zoom and dual goosenecks to supply oblique illumination; CO2 equipped fly sorting station (Leica, model: MZ16 ; custom fabrication)
    2. Forceps, Dumont #5 (Fine Science Tools, catalog number: 11252-30 )
    3. Dissecting dish (Thermo Fisher Scientific, Fisher Scientific, catalog number: 21-379 )
    4. FACS Aria II sorter (BD)

  2. RNA isolation
    1. Vortex

  3. Library construction
    1. DynaMagTM-2 magnet (Thermo Fisher Scientific, catalog number: 12321D )
    2. Agencourt AMPure XP 5 ml Kit (Beckman Coulter, catalog number: A63880 )
    3. PCR thermal cycler
    4. Qubit® 3.0 Fluorometer (Thermo Fisher Scientific, InvitrogenTM, model: Qubit® 3.0 Fluorometer)
    5. Agilent 2100 Bioanalyzer (Agilent Technologies, model: 2100 Bioanalyzer)
    6. Applied BiosystemsTM 7500 Fast & 7500 real-time PCR system (Thermo Fisher Scientific, model: 7500 Fast & 7500 real-time PCR system)
    7. Hiseq-2500 sequencing system (Illumina, model: Hiseq-2500)

Software

  1. Mapping and analysis of Illumina reads for transcriptome
    1. Bowtie (http://bowtie-bio.sourceforge.net/index.shtml, Langmead et al., 2009)
    2. TopHat (http://ccb.jhu.edu/software/tophat/index.shtml, Trapnell et al., 2009)
    3. Cufflinks (http://cole-trapnell-lab.github.io/cufflinks/, Roberts et al., 2011)
    4. CASAVA (http://support.illumina.com.cn/sequencing/sequencing_software/casava.html, Illumina)

Procedure



Figure 1. Protocol overview. Under RNase-free environment, the midguts with foregut and hindgut portion removed (left panel) are digested with Elastase for 1 h. The dissociated tissues are centrifuged, resuspended in DEPC-PBS, filtered, and then sorted through FACS. About 250,000 sorted cells are collected to harvest total RNA, which is then used for library construction and sequencing with Illumina Hiseq-2500 system. The upper right panel shows a confocal image of midgut epithelium with Esg-GFP expression. The bottom right panel highlights the Esg-GFP+ cell population measured by FACS.

  1. Isolation of intestinal progenitor cells by FACS (Figure 1)
    1. The fly strains carrying Esg-GFP fluorescent markers are used to sort intestinal progenitor cell population (including intestinal stem cells and enteroblasts), midguts from w1118 strain are used to set fluorescence gate. To achieve 250K Esg-GFP+ cells, a minimum of 1,000 midguts is required.
      Note: It is important to be consistent with age, gender and culture conditions among different biological samples. Here we take expression analysis of Sox21a mutant flies for example. Sox21a mutants carrying Esg-GFP markers served as the mutant sample, with Esg-GFP wild-type flies as WT Ctrl. Based on the observation that overproliferation phenotype is displayed after 10 days in 90% sox21a mutant intestines, we analyzed flies at 10 days old for both the mutants and WT control. For both groups, we chose female flies and cultured them with identical food (standard fly food plus yeast paste).
    2. The following steps are performed under RNase-free conditions. Forceps and the dissecting pads are pre-washed with DEPC-PBS solution. The prepared females are ice-anesthetized for dissection. Intestines are dissected and the foregut and hindgut parts are removed by forceps (Figure 2). The midguts are then immediately put in DEPC-PBS (see Recipes) solution on ice.


      Figure 2. The midgut dissection procedure. A. Use forceps to gently hold a fly and tear the abdomen at the boundary of thorax/abdomen, pull the abdomen away from the anterior part without touching the gut. B and C. Before the gut is fully stretched, cut the gut at the boundaries between foregut and hindgut. Remove the appendix if Malpighian tubule (Mt) or ovarium is attached to the midgut.

    3. Incubate 100-200 guts with 1 mg/ml Elastase (see Recipes) in about 1 ml DEPC-PBS per microcentrifuge tube for 1 h at 25 °C until the midguts are largely dissociated. Softly mix the sample every 15 min by pipetting and inverting several times.
    4. Dissociated samples are pelleted at 400 x g for 20 min, and resuspended in a microcentrifuge tube with 0.5 ml DEPC-PBS. The suspension was filtered with 40 or 70-μm filters (Corning), by touching pipette tips on the top of the filter so that the cells can go through filters. Wash the tubes and the filters with 0.5 ml DEPC-PBS, and also collect the filtered suspension in the filtered cells. The filtered cells are then sorted using a FACS Aria II sorter (BD Biosciences) and collected in 0.5 ml DEPC-PBS.
      Note: 40-μm filters are recommended for pure isolation of small diploid cells, such as intestinal progenitor cells or enteroendocrine cells. It’s recommended to double check the sorted cells based on cell morphology and expression of the fluorescent marker.
    5. The sorted cells are pelleted at 400 x g for 20 min and preserved in 0.2-0.8 ml RNAiso Plus (Takara Bio) reagents, which can be stored at -80 °C within 6 months until RNA isolation. For each of the biological replicates, at least 200,000 sorted cells are collected to harvest total RNA. For each genotype group, at least 3 biological replicates are prepared.

  2. RNA isolation
    1. Add equal volume of > 95% ethanol directly to the homogenate cell sample. Mix thoroughly by vortexing.
    2. Follow the Directzol RNA MiniPrep Kit to harvest total RNA of each sample. DNase I treatment is unnecessary, as polyA RNAs will be selectively purified during cDNA library construction. The RNA sample eluted in DNase/RNase-free water should be used immediately for sequencing library construction or stored at -80 °C for up to 3 months.

  3. Library construction
    1. Qualify total RNA on Agilent 2100 Bioanalyzer using Agilent RNA 6000 Pico Kit. Purify polyA RNA from 50 ng total RNA using Oligo-dT Dynabeads according to manufacturer’s protocol (Dynabeads® mRNA DIRECTTM Purification Kit) and elute the polyA RNA in 12 μl RNase free water.
    2. RNA fragmentation
      PolyA RNA                                                                        12 μl
      5x first strand buffer                                                      6 μl
      Incubate at 95 °C 5 min and chill on ice
    3. Reverse transcription
      Add 1.5 μl Hexadeoxyribonucleotide mixture (50 ng/μl) to each sample, incubate at 65 °C for 5 mins and chill on ice.
      Prepare the following RT mix on ice (1x):
      dNTP mixture (10 mM)                                                    1.5 μl
      DTT (0.1 M)                                                                      3 μl
      Ribonuclease inhibitor (40 U/μl)                                   1 μl
      SuperScript II Reverse Transcriptase (200 U/μl)         1 μl
      Nuclease-free water                                                         4 μl
      Add 10.5 µl of the RT mix to each sample, mix and spin. Transfer to thermocycler:
      25 °C 10 min
      42 °C 50 min
      70 °C 15 min
      4 °C hold
    4. Synthesis second strand of cDNA
      First strand cDNA                                                        30 μl
      dNTP mixture (10 mM)                                                2 μl
      Second strand buffer (5x)                                           10 μl
      RNase H (5 U/μl)                                                         1 μl
      DNA polymerase I (5 U/μl)                                          3 μl
      Nuclease-free water                                                    4 μl
      16 °C 2.5 h
      4 °C hold
      Purify cDNA using 1.8x AMPure XP beads, and elute in Nuclease-free water.
    5. Construct cDNA library using NEBNex® DNA library prep master mix set and NEBNext® multiplex oligos.
    6. Qualify library on Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA Kit. Quantify library using Qubit® dsDNA HS Assay Kit and Illumina Library Quantification Kit according to manufacturer’s protocol. Run library on the Illumina Hiseq-2500 sequencing system.

Data analysis

  1. Mapping and analysis of Illumina reads for transcriptome
    1. Illumina Casava1.8 software used for basecalling.
    2. Drosophila melanogaster genome sequences and bowtie index can be downloaded from Tophat website (Figure 3).


      Figure 3. Screenshots of the Tophat webpage

    3. Single-end reads are mapped to the Drosophila melanogaster genome (Release 5) using TopHat (v2.0.10). For our analysis, we allow up to 2 mismatches and aligned with command ‘tophat --bowtie1 –N 2 --no-coverage-search –G genes.gtf genome sample.fastq’, where genes.gtf is the structure of genes and sample.fastq is the raw sequencing reads of a sample.
    4. Cuffquant is used for quantifying gene and transcript expression levels for a sample BAM file with command ‘cuffquant -o outputdir genes.gtf -p 15 sample_aligned/accepted_hits.bam’. This step generates .cxb files which contain gene and transcript expression levels.
    5. We use cuffnorm for computing normalized expression values (FPKM) of genes of all samples. csDendro and MDSplot are used for provide insight into the relationships between replicates and different conditions. This step makes sure the replicates samples are consistent and can be used for subsequent analysis (Figure 4).
    6. Differentially expressed genes are identified by cuffdiff and significant differentially expression genes were filtered with P value ≤ 0.05, and fold change ≥ 2.


      Figure 4. csDendro (left) and MDSplot (right) are used to evaluate the relationships among experimental and control groups. Here the example shows 3 replicates of sox21a mutant samples (Sox21a-1, -2, -3) and 4 replicates of WT control samples (WT-1, -2, -3, -4).

Recipes

  1. 1x DEPC-PBS
    1x PBS solution (pH 7.2-7.4) contains 137 mM NaCl, 2.7 mM KCI, 4.3 mM Na2HPO4 and 1.4 mM KH2PO4
    0.1% final solution of diethyl pyrocarbonate (DEPC) is added to 1x PBS
    Mix well by shaking and leave overnight in a fume hood at room temperature
    The solution can be stored at room temperature up to a year in RNase-free conditions
  2. Elastase solution
    Dissolve elastase in 1x DEPC-PBS buffer at a final concentration of 1 mg/ml

Acknowledgments

Sorting of intestinal progenitor cells was performed according to the method described previously (Dutta et al., 2015) with some modifications. This work was supported by National Basic Science 973 grant (2014CB850002) from the Chinese Ministry of Science and Technology.

References

  1. Chen, J., Xu, N., Huang, H., Cai, T. and Xi, R. (2016). A feedback amplification loop between stem cells and their progeny promotes tissue regeneration and tumorigenesis. Elife 5. pii: e14330
  2. Dutta, D., Buchon, N., Xiang, J. and Edgar, B. A. (2015). Regional cell specific RNA expression profiling of FACS isolated Drosophila intestinal cell populations. Curr Protoc Stem Cell Biol 34: 2F 2 1-14.
  3. Langmead, B., Trapnell, C., Pop, M. and Salzberg, S. L. (2009). Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10(3): R25.
  4. Trapnell, C., Pachter, L. and Salzberg, S. L. (2009). TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25(9): 1105-1111.
  5. Roberts, A., Trapnell, C., Donaghey, J., Rinn, J. L. and Pachter, L. (2011). Improving RNA-Seq expression estimates by correcting for fragment bias. Genome Biol 12(3): R22. 

简介

RNA测序(RNA-seq)已经成为描述基因表达的流行方法。 在许多应用中,一个共同目的是在不同的生物学或病理学条件下鉴定差异表达的基因和途径。 该方案提供了〜250,000个分选的果蝇肠细胞的RNA-seq分析的详细程序(Chen等,2016),其中不需要RNA扩增。
【背景】RNA-seq的转录组分析已经成为鉴定不同生物或病理条件下差异表达基因和途径的常用方法。 对于产生低mRNA水平的样品,通常在深度测序之前进行RNA或cDNA扩增(Dutta等,2015)。 然而,这个程序可能会潜在地省略以低丰度表达的重要候选人。 在这里,我们提供了不需要RNA扩增的分选的果蝇肠细胞的RNA-seq分析的详细程序。

关键字:RNA-seq, FACS, 果蝇肠道, 祖细胞, 转录组分析

材料和试剂

  1. 通过FACS分离肠祖细胞
    1. 微量离心管(Corning,Axygen ®,目录号:MCT-150-C)
    2. 40微米过滤器
    3. 70μm过滤器(Corning,Falcon ®,目录号:352350)
    4. 携带Esg-GFP荧光标记的蝇株
    5. 弹性蛋白酶(Sigma-Aldrich,目录号:E0258)
    6. RNAiso Plus(Takara Bio,目录号:9108/9109)
    7. NaCl
    8. KCI
    9. Na 2 HPO 4
    10. KH 2 PO 4
    11. 焦碳酸二乙酯(DEPC)(Sigma-Aldrich,目录号:D5758)
    12. 1x DEPC-PBS(参见食谱)
    13. 弹性蛋白酶溶液(参见食谱)

  2. RNA分离
    1. Directzol RNA MiniPrep试剂盒(ZYMO RESEARCH,目录号:R2050)
    2. 95-100%乙醇

  3. 图书馆建设
    1. Agilent RNA 6000 Pico Kit(Agilent Technologies,目录号:5067-1513)
    2. Dynabeads ® mRNA DIRECT TM纯化试剂盒(Thermo Fisher Scientific,Ambion TM,目录号:61011)
    3. 六溴代核糖核苷酸混合物,pd(N)6(Takara Bio,目录号:3801)
    4. dNTP混合物(Takara Bio,目录号:4019)
    5. DTT(由SuperScript提供 II逆转录酶)
    6. 重组RNasin 核糖核酸酶抑制剂(Promega,目录号:N2511)
    7. SuperScript ® II逆转录酶(Thermo Fisher Scientific,Invitrogen TM,目录号:18064014)
    8. 第二链缓冲液(Thermo Fisher Scientific,Invitrogen TM,目录号:10812014)
    9. DNA聚合酶I(Takara Bio,目录号:2130A)
    10. RNase H(New England Biolabs,目录号:M0297S)
    11. Agencourt AMPure XP Kit(5 ml)(Beckman Coulter,目录号:A63880)
    12. NEBNext ®用于Illumina (New England Biolabs,目录号:E6040L)的DNA文库准备主混合组
    13. NEBNext ®用于Illumina的多重寡核苷酸(New England Biolabs,目录号:E7335S/E7500S)
    14. 安捷伦高灵敏度DNA试剂盒(Agilent Technologies,目录号:5067-4626)
    15. Qubit ® dsDNA HS测定试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32851)
    16. Illumina Library Quantification Kit(Kapa Biosystems,目录号:KK4824)

设备

  1. 通过FACS分离肠祖细胞
    1. 用变焦和双鹅颈管解剖显微镜供应斜照射; CO 2配备飞行分拣站(Leica,型号:MZ16;定制)
    2. 镊子,Dumont#5(精细科学工具,目录号:11252-30)
    3. 解剖盘(Thermo Fisher Scientific,Fisher Scientific,目录号:21-379)
    4. FACS Aria II分拣机(BD)

  2. RNA分离
    1. 涡流

  3. 图书馆建设
    1. DynaMag TM -2磁体(Thermo Fisher Scientific,目录号:12321D)
    2. Agencourt AMPure XP 5 ml试剂盒(Beckman Coulter,目录号:A63880)
    3. PCR热循环仪
    4. Qubit ® 3.0荧光计(Thermo Fisher Scientific,Invitrogen TM,型号:Qubit 3.0荧光计)
    5. Agilent 2100生物分析仪(Agilent Technologies,型号:2100 Bioanalyzer)
    6. Applied Biosystems TM 7500 Fast& 7500实时PCR系统(Thermo Fisher Scientific,型号:7500 Fast& 7500实时PCR系统)
    7. Hiseq-2500测序系统(Illumina,型号:Hiseq-2500)

软件

  1. 转录组的Illumina读数的绘制和分析
    1. Bowtie( http://bowtie-bio.sourceforge.net/index。 shtml ,Langmead 等人,2009)
    2. TopHat( http://ccb.jhu.edu/software/tophat/index.shtml ,Trapnell等,2009)
    3. 袖扣( http://cole-trapnell-lab.github.io/cufflinks/,Roberts等人,2011)
    4. CASAVA( http://support.illumina.com。 cn/sequencing/sequencing_software/casava.html ,Illumina)

程序



图1.协议概述。 在不含RNase的环境中,除去前肠和后肠部分的中肠(左图)用Elastase消化1小时。将离解的组织离心,重悬于DEPC-PBS中,过滤,然后通过FACS分选。收集约250,000个分选的细胞以收获总RNA,然后将其用于文库构建和用Illumina Hiseq-2500系统测序。右上图显示了具有Esg-GFP表达的中肠上皮的共焦图像。右下方的面板突出显示了通过FACS测量的Esg-GFP + 细胞群体。

  1. 通过FACS分离肠祖细胞(图1)
    1. 携带Esg-GFP荧光标记的蝇株用于分选肠祖细胞群(包括肠干细胞和成肠细胞),使用来自w111018菌株的中肠设置荧光门。要实现250K的Esg-GFP + 细胞,需要至少1,000个中肠。
      注意:与不同生物样本中的年龄,性别和文化条件保持一致很重要。这里我们以Sox21a突变体蝇为例进行表达分析。携带Esg-GFP标记的Sox21a突变体作为突变体样品,Esg-GFP野生型苍蝇作为WT Ctrl。基于在90%的sox21a突变体肠中10天后显示过度增殖表型的观察,我们分析了10天龄的突变体和WT对照的苍蝇。对于这两个组,我们选择女性苍蝇,并用相同的食物(标准飞行食物加酵母膏)进行培养
    2. 在无RNase的条件下进行以下步骤。镊子和解剖垫用DEPC-PBS溶液预洗。准备的女性被冰麻醉以进行解剖。解剖肠,并用镊子去除前肠和后肠部分(图2)。然后将中肠立即放入DEPC-PBS(参见食谱)溶液在冰上。


      图2.中肠解剖程序 A.使用镊子轻轻握住一只苍蝇,撕开胸部/腹部边界的腹部,将腹部从前部拉出,而不用接触肠道。 B和C.在肠完全拉伸之前,切除前肠和后肠之间的肠道。如果马鞭状小管(Mt)或卵巢附着在中肠,请移除附件。

    3. 使用1毫克/毫升弹性蛋白酶(参见食谱)将100-200肠内注入约1ml DEPC-PBS /微量离心管中,在25℃下孵育1小时,直至中肠大部分解离。每15分钟轻轻搅拌一次,抽取和倒置数次。
    4. 分离的样品在400×g下沉淀20分钟,并且用0.5ml DEPC-PBS重悬于微量离心管中。用40或70-μm过滤器(Corning)过滤悬浮液,通过接触过滤器顶部的移液管吸头,使细胞通过过滤器。用0.5ml DEPC-PBS洗涤管和过滤器,并且还将过滤的悬浮液收集在过滤的细胞中。然后使用FACS Aria II分选机(BD Biosciences)对经过滤的细胞进行分选,并收集在0.5ml DEPC-PBS中。
      注意:建议使用40μm过滤器纯化分离小二倍体细胞,如肠祖细胞或肠内分泌细胞。建议根据细胞形态和荧光标记物的表达来仔细检查分选的细胞。
    5. 分选的细胞在400×g下沉淀20分钟并保存在0.2-0.8ml RNAiso Plus(Takara Bio)试剂中,其可以在6个月内在-80℃下储存直到RNA分离。对于每个生物重复,收集至少20万个分选的细胞以收获总RNA。对于每个基因型组,至少制备3个生物重复
  2. RNA分离
    1. 加入等体积的> 95%乙醇直接用于匀浆细胞样品。通过涡旋彻底混合。
    2. 按照Directzol RNA MiniPrep Kit收集每个样品的总RNA。 DNA酶I处理是不必要的,因为polyA RNA将在cDNA文库构建期间被选择性纯化。在DNA酶/无RNA酶水中洗脱的RNA样品应立即用于测序文库构建,或在-80°C下储存长达3个月。

  3. 图书馆建设
    1. 使用Agilent RNA 6000 Pico Kit对Agilent 2100生物分析仪上的总RNA进行鉴定。使用Oligo-dT Dynabeads根据制造商的方案(Dynabeads mRNA DIRECT TM纯化试剂盒)纯化来自50ng总RNA的polyA RNA,并以12μl无RNase的方式洗脱polyA RNA水
    2. RNA片段化
      PolyA RNA                                                                    12μl
      5x第一线缓冲区                             ;                          ;     6μl
      在95°C下孵育5分钟,在冰上冷却
    3. 逆转录
      向每个样品中加入1.5μl己内酰氧基核糖核苷酸混合物(50ng /μl),在65℃孵育5分钟,并在冰上冷却。
      在冰(1x)上准备以下RT组合:
      dNTP混合物(10 mM)                                                 1.5μl
      DTT(0.1M)                                                                    3μl
      核糖核酸酶抑制剂(40 U /μl)                                    1μl
      SuperScript II逆转录酶(200 U /μl)         1μl
      无核酸酶水                        ;                          ;     4μl
      向每个样品中加入10.5μlRT混合物,混合并旋转。转移到热循环仪:
      25°C 10分钟
      42℃50分钟
      70°C 15分钟
      4℃保持
    4. 合成第二链cDNA 第一串cDNA                                                          30μl
      dNTP混合物(10 mM)                                                2μl
      第二线缓冲器(5x)                                           10μl
      RNase H(5 U /μl)                                                            1μl
      DNA聚合酶I(5 U /μl)                      ;                        3μl
      无核酸酶水                        ;                          ;    4μl
      16℃2.5小时
      4℃保持
      使用1.8x AMPure XP珠纯化cDNA,并在无核酸酶的水中洗脱。
    5. 使用NEBNex DNA文库制备主混合组和NEBNext多重寡核苷酸构建cDNA文库。
    6. 使用安捷伦高灵敏度DNA试剂盒对Agilent 2100生物分析仪进行鉴定。根据制造商的方案使用Qubit ® dsDNA HS测定试剂盒和Illumina Library定量试剂盒定量文库。在Illumina Hiseq-2500测序系统上运行库。

数据分析

  1. 用于转录组的Illumina读取的映射和分析
    1. Illumina Casava1.8软件,用于基准测试。
    2. 基因组序列和bowtie指数可以从Tophat网站下载(图3)。


      图3. Tophat网页的屏幕截图

    3. 使用TopHat(v2.0.10)将单端读取映射到黑腹果蝇基因组(版本5)。对于我们的分析,我们允许多达2个不匹配,并与命令'tophat --bowtie1 -N 2 - 无覆盖搜索-G基因gt基因组sample.fastq',其中,gene.gtf是基因的结构, sample.fastq是样本的原始排序读取。
    4. Cffquant用于定量样品BAM文件的基因和转录表达水平,命令为'cuffquant -o outputdir genes.gtf -p 15 sample_aligned/accepted_hits.bam'。此步骤生成含有基因和转录表达水平的.cxb文件
    5. 我们使用cuffnorm计算所有样品的基因的标准化表达值(FPKM)。 csDendro和MDSplot用于提供对复制和不同条件之间关系的了解。此步骤确保重复样本是一致的,并可用于后续分析(图4)
    6. 差异表达基因通过袖扣鉴定,并且显着的差异表达基因被筛选为≥0.05,倍数变化≥2。


      图4. csDendro(左)和MDSplot(右)用于评估实验组和对照组之间的关系。此示例显示了3个重复的sox21a突变体样品(Sox21a -1,-2,-3)和WT对照样品(WT-1,-2,-3,-4)的4个重复。

食谱

  1. 1x DEPC-PBS
    1×PBS溶液(pH7.2-7.4)含有137mM NaCl,2.7mM KCl,4.3mM Na 2 HPO 4和1.4mM KH 2 PO 4
    将0.1%焦碳酸二乙酯(DEPC)的最终溶液加入到1×PBS中 通过振荡混合,并在室温下在通风橱中离开过夜 该解决方案可以在无RNase的条件下在室温下储存多达一年
  2. 弹性酶溶液
    将1%DEPC-PBS缓冲液中的弹性蛋白酶溶解至终浓度为1 mg/ml

致谢

根据先前描述的方法(Dutta等人,2015)对一些肠祖细胞进行分类,并进行一些修改。这项工作得到了中国科学技术部"国家基础科学973"(2014CB850002)的支持。

参考文献

  1. Chen,J.,Xu,N.,Huang,H.,Cai,T. and Xi,R。(2016)。  干细胞及其后代之间的反馈扩增回路促进组织再生和肿瘤发生。 5.ii:e14330 < br />
  2. Dutta,D.,Buchon,N.,Xiang,J.和Edgar,B.A。(2015)。 分离的FACS的FACS的区域细胞特异性RNA表达谱分析<肠细胞群体。 Curr Protoc Stem Cell Biol 34:2F 2 1-14。
  3. Langmead,B.,Trapnell,C.,Pop,M。和Salzberg,S.L。(2009)。 将短DNA序列与人类基因组进行超快速记忆有效的比对。基因组生物 10(3):R25。
  4. Trapnell,C.,Pachter,L。和Salzberg,SL(2009)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/19289445"target ="_ blank"> TopHat:发现具有RNA-Seq的剪接点。生物信息学25(9):1105-1111。
  5. Roberts,A.,Trapnell,C.,Donaghey,J.,Rinn,J.L。和Pachter,L。(2011)。 通过纠正片段偏差来改进RNA-Seq表达估计。基因组生物 12(3):R22。
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Copyright Chen et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Chen, J., Li, J., Huang, H. and Xi, R. (2016). Gene Expression Analysis of Sorted Cells by RNA-seq in Drosophila Intestine. Bio-protocol 6(24): e2079. DOI: 10.21769/BioProtoc.2079.
  2. Chen, J., Xu, N., Huang, H., Cai, T. and Xi, R. (2016). A feedback amplification loop between stem cells and their progeny promotes tissue regeneration and tumorigenesis. Elife 5. pii: e14330
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