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Mar 2018

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In planta Transcriptome Analysis of Pseudomonas syringae
丁香假单胞菌的植物转录组分析   

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Abstract

Profiling bacterial transcriptome in planta is challenging due to the low abundance of bacterial RNA in infected plant tissues. Here, we describe a protocol to profile transcriptome of a foliar bacterial pathogen, Pseudomonas syringae pv. tomato DC3000, in the leaves of Arabidopsis thaliana at an early stage of infection using RNA sequencing (RNA-Seq). Bacterial cells are first physically isolated from infected leaves, followed by RNA extraction, plant rRNA depletion, cDNA library synthesis, and RNA-Seq. This protocol is likely applicable not only to the A. thaliana–P. syringae pathosystem but also to different plant-bacterial combinations.

Keywords: RNA-Seq (转录组测序技术), Bacterial pathogen (细菌性病原体), Arabidopsis (拟南芥), Transcriptome (转录组), Pseudomonas (假单胞菌)

Background

Plants have evolved innate immune systems to fend off pathogen attack. Molecular mechanisms of pathogen recognition and immune signaling pathways have been intensively studied over the past decades. However, how plant immunity affects pathogen metabolism to inhibit pathogen growth is scarcely understood often because of the difficulty in profiling pathogen responses in planta. In case of bacterial pathogens, transcriptome profiling inside plant leaves is difficult to study because the amount of bacterial mRNA is much lower than that of plants; and it is particularly challenging at an early stage of infection due to the low population density of bacteria in plants. To overcome this limitation, we established a method for isolating bacteria from the infected plant leaves and profiling bacterial transcriptome with RNA-Seq. This method has been successfully used for profiling the transcriptome of the model bacterial pathogen Pseudomonas syringae pv. tomato DC3000 in the model plant Arabidopsis thaliana in various conditions (Nobori et al., 2018).

Materials and Reagents

  1. 50 ml Falcon tubes (Corning, Falcon®, catalog number: 352070 )
  2. 1.5 ml Eppendorf Safe-Lock tubes (Eppendorf, catalog number: 0030120086 )
  3. Pipette tips, 1,000 μl and 10 μl volumes (Corning, DeckWorksTM, catalog numbers: 4124 and 4120 )
  4. Sterile Serological Pipet, 25 ml (Corning, catalog number: 4251 )
  5. 1 ml needleless syringe (BD, Luer-LokTM, catalog number: 303172 )
  6. 4 mm stainless steel balls (Mühlmeier)
  7. 6 μm filter mesh (Bückmann, catalog number: 20000796 )
  8. 30-32 days old Arabidopsis thaliana plants
  9. Pseudomonas syringae pv. tomato DC3000 (Pto)
  10. 100% ethanol (VWR International, catalog number: 20821.321 )
  11. Chloroform (Carl Roth, catalog number: 4432 )
  12. peqGOLD TriFast (VWR, PeqLab, catalog number: 30-2010 )
  13. Phenol solution pH 8.0 (Sigma-Aldrich, catalog number: P4557 )
  14. Nuclease-Free Water (not DEPC-Treated) (Thermo Fisher Scientific, InvitrogenTM, Ambion®, catalog number: AM9932 )
  15. Tris(2-carboxyethyl)phosphine (TCEP) (Sigma-Aldrich, catalog number: C4706 )
  16. RNAqueous®-Micro Total RNA Isolation Kit (Thermo Fisher Scientific, InvitrogenTM, Ambion®, catalog number: AM1931 )
  17. TURBO DNA-free Kit (Thermo Fisher Scientific, InvitrogenTM, Ambion®, catalog number: AM1907 )
  18. Ribo-Zero rRNA Removal Kit (Plant) (Illumina, catalog number: MRZPL1224 )
  19. RNeasy Mini Kit (QIAGEN, catalog number: 74104 )
  20. Ovation® Complete Prokaryotic RNA-Seq DR Multiplex Systems (NuGEN Technologies, catalog number: 0326-32 )
  21. QIAGEN-tip 500 (QIAGEN, catalog number: 10063 )
  22. Liquid nitrogen
  23. NaOH (Carl Roth, catalog number: 6771 )
  24. Bacterial isolation buffer (see Recipes)

Equipment

  1. Eppendorf Reference Pipette (Eppendorf, 100-1,000 μl)
  2. Gilson PIPETMAN Classic pipette (Gilson, model: P20, catalog number: F123600 )
  3. Electric pipette (BrandTech Scientific, model: accu-jet® pro, catalog number: 26330 )
  4. Refrigerated centrifuge (Eppendorf, models: 5417 R and 5810 R )
  5. Roller mixer SRT1 (Stuart Scientific, model: SRT1 )
  6. Vortex mixer (Scientific Industries, model: Vortex-Genie 2 , catalog number: SI-0236)
  7. Shaker
  8. Thermal cycler (DNA Engine Tetrad 2 Peltier Thermal Cycler, Bio-Rad, model: Tetrad 2 )
  9. NanoDropTM One/OneC Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM OneC , catalog number: ND-ONE-W)
  10. -80 °C freezer
  11. Autoclave
  12. Fume hood
  13. Forceps (Fine Science Tools, model: Dumont #5, catalog number: 11251-10 )
  14. Plastic cutter
  15. Scissors
  16. Illumina HiSeq-3000 sequencer

Software

  1. BowTie2
  2. Python package HTSeq

Procedure

The overview of the procedure is shown in Figure 1.


Figure 1. Workflow summary of in planta bacterial transcriptome analysis. The figure is adopted from Nobori et al. (2018), PNAS. Leaves infected with P. syringae are crushed and incubated in the bacterial isolation buffer, followed by filtering out large plant tissues. Then the flow through is centrifuged to separate bacterial cells from plant cells. Bacterial cells are harvested and subjected to RNA extraction, cDNA library preparation, and RNA-seq.


  1. Plant growth condition
    Plants were grown in a chamber at 22 °C with a 10-h light period and 60% relative humidity for 24 d and then in another chamber at 22 °C with a 12-h light period and 60% relative humidity. Light intensity was around 120 μmol/m2/sec. Thirty to thirty-two days old A. thaliana plants old were used.

  2. Bacterial isolation (Day 1)
    Note: Use protective equipment while touching the frozen tubes.
    1. Infiltrate bacterial suspension (OD600 = 0.5) to A. thaliana leaves (4 leaves/plant x 20 plants, 80 leaves in total, approximately 3 g) from the abaxial side with a 1 ml needle-less syringe. Make sure the entire leaf area infiltrated with the bacteria.
    2. Six hours after infiltration, collect the 80 leaves with forceps into a 50 ml Falcon tube containing 8-10 stainless steel balls (4 mm; sterilized by washing with ethanol and air dried). Flash-freeze with liquid nitrogen. Samples can be stored at -80 °C.
    3. Shake thoroughly by hand and crush the leaves into small pieces (Figure 2). Ensure that the samples do not thaw during crushing.


      Figure 2. Crushed leaf sample before incubation in the bacterial isolation buffer

    4. Add 30 ml ice-cold bacterial isolation buffer in a fume hood and vortex immediately for 10 sec followed by shaking vigorously by hand for 10 sec. Incubate the tubes for 20 h with shaking at 33 rpm using Roller mixer SRT1 at 4 °C.

  3. Bacterial isolation (Day 2)
    Note: Steps C1-C7 and D1-D12 should be done in a fume hood. 
    1. Equip a 50 ml Falcon tube with a filter holder (handmade from a QIAGEN-tip 500 column; the upper half was cut by a plastic cutter; autoclaved before using) and a single layer of 6 µm filter mesh (cut into 12 cm x 15 cm size, and then autoclaved) (Figure 3). Trim away the excess filter mesh with scissors so that the cap can fit the tube.


      Figure 3. Filter and filter holder equipped to a 50 ml Falcon tube. A. A QIAGEN-tip 500 column is cut to make a filter holder. B. A 50 ml Falcon tube, a 6 µm filter mesh (12 cm x 15 cm), and the filter holder. C. Equip the 50 ml Falcon tube with the filter holder and the filter mesh. D. Trim away the excess filter mesh so that the cap can fit the tube. 

    2. Apply the sample (15 ml; without stainless steel balls) to the column and filter it by centrifuging at 1,300 x g for 10 sec (or until all the liquid goes through) at 4 °C. While keeping the flow-through in the tube, apply the rest of the sample (15 ml) to the column and repeat the filtering.
    3. Remove the filter and the filter holder. Centrifuge the flow-through for 20 min at 3,200 x g at 4 °C. Discard the supernatant with an electronic pipette equipped with a 25 ml serological pipette. You may leave some liquid (100 μl) to avoid the sample loss.
    4. Add 900 μl of ice-cold bacterial isolation buffer and resuspend the pellet by vortexing, then transfer it to a new 1.5 ml tube on ice by pipetting. Make sure that the pellet is completely resuspended.
    5. Centrifuge for 20 min at 2,300 x g at 4 °C. You will see two-layered pellet: white on the top and green at the bottom (Figure 4).
      Note: Sometimes the upper part of the tube gets pale green. In this case, you may remove the green part by pipetting with a small amount of the liquid phase and discard it to avoid a potential plant contamination.


      Figure 4. A two-layered pellet consists of plants (bottom, green) and bacteria (top, white)

    6. Equip 20 μl Gilson pipette with 10 μl filter tip (with 20 μl capacity) and set the volume to 18 μl. Carefully resuspend only the top white layer by pipetting up and down close to the surface of the pellet. When the buffer gets cloudy by the bacterial cells, transfer the buffer (approximately 1,000 μl) to a new 1.5 ml tube on ice using 1,000 μl pipette. Then, carefully add 1 ml of new ice-cold bacterial isolation buffer without disrupting the pellet by tiling the tube. Repeat resuspending and collecting bacterial cell suspensions in new 1.5 ml tubes (total three times or until the white layer is completely removed).
      Notes:
      1. You may use larger tips that fit the pipette, but smaller tips make it easier to resuspend the bacterial layer without breaking the plant layer.
      2. If the layer of plant tissues is collapsed and mixed with the layer of bacterial cells, you can recover the bacterial layer by completely resuspending the entire pellet by vortexing and then repeating the centrifugation step (Step C5).
    7. Centrifuge the bacterial suspensions for 2 min at 10,000 x g to harvest bacterial cells. Discard the supernatant by pipetting.

  4. RNA extraction
    1. Add 1 ml of peqGOLD TriFast per sample to the pellets. For example, if you have 3 tubes after Step B6, add 333 μl of peqGOLD TriFast to each tube, vortex, and merge them in one tube. Samples can be stored at -80 °C.
    2. Add 200 μl of chloroform per 1 ml of peqGOLD TriFast, vortex, and leave the tube for 2 min at room temperature (RT).
    3. Centrifuge for 15 min at 12,000 x g at 4 °C. Take the aqueous part (typically 400-500 μl) into a new 1.5 ml Eppendorf tube by pipetting.
    4. Isolate RNA using RNAqueous®-Micro Total RNA Isolation Kit (Invitrogen, Ambion®) according to the manufacturer's protocol with some modifications (see below).
    5. Preheat Elution Solution to 75 °C.
    6. Add half-volume of 100% ethanol to the sample and vortex briefly.
    7. Load the sample (up to 150 μl) onto a Micro Filter Cartridge Assembly. Centrifuge for 10 sec at 12,000 x g at RT. Repeat this step until all of the samples are loaded. Discard flow-through.
    8. Add 180 μl of Wash Solution 1 to the Micro Filter Cartridge. Centrifuge for 10 sec at 12,000 x g at RT.
    9. Add 180 μl of Wash Solution 2/3 to the Micro Filter Cartridge. Centrifuge for 10 sec at 12,000 x g at RT.
    10. Repeat Step C9 one more time.
    11. Replace the Micro Filter Cartridge into a fresh Collection Tube. Centrifuge for 1.5 min at 12,000 x g at RT to remove residual fluid and dry the filter.
    12. Replace the Micro Filter Cartridge into a new 1.5 ml Eppendorf tube. Elute RNA with 15 μl of preheated Elution Solution. Repeat elution one more time into the same tube (30 μl in total).
    13. Perform DNase treatment with TURBO DNA-free kit (Ambion®) according to the manufacturer's protocol.
    14. Check the RNA concentration with NanoDrop. Typical RNA concentration is 100-200 ng/μl.

  5. Plant-derived rRNA depletion
    Follow the protocol of Ribo-Zero rRNA Removal Kit (Plant). Use RNeasy Mini Kit (QIAGEN) for RNA purification as described in the manufacturer's protocol. Typical RNA yield is 100-400 ng.

  6. Library preparation and sequencing
    1. cDNA library is synthesized using Ovation® Complete Prokaryotic RNA-Seq DR Multiplex Systems (NuGEN) according to the manufacturer's protocol with some modifications.
      IMPORTANT: Input 10 ng of bacteria-enriched RNA, although the kit recommends 100-500 ng. This is because you cannot always obtain this much enriched RNA. Keep the RNA input amount consistent.
    2. Sequence the library with an Illumina HiSeq sequencer.

Data analysis

Mapping and counting of Illumina reads for transcriptome:

  1. Sequence reads were mapped on Pto genome (available at http://pseudomonas.com/) using BowTie2 (Langmead and Salzberg, 2012). Sequence reads with the phred quality score greater than 33 were taken as an input (Command: --phred33). We use default settings for the other parameters.
  2. Mapped reads were counted with the Python package HTSeq (Anders et al., 2015) with default settings. The gene annotation file (GFF3 file) of Pto is available at http://pseudomonas.com/.

Notes

  1. Both phenol and chloroform are toxic. Handle them in a fume hood and wear proper protection.
  2. In Step D14, the quality of RNA tends to be variable and often low. However, this did not cause problems in data reproducibility and accuracy of RNA-Seq measurement in our previous experiments (Nobori et al., 2018).

Recipes

  1. Bacterial isolation buffer
    Nuclease-Free Water
    25 mM TCEP (tris(2-carboxyethyl)phosphine), pH 4.5 adjusted with 10 N NaOH
    9.5% Ethanol
    0.5% Phenol solution
    Note: Prepare it fresh every time.

Acknowledgments

This protocol has been adapted from a previously published paper (Nobori et al., 2018). This work was supported by the Max Planck Society and Deutsche Forschungsgemeinschaft Grant SFB670 (to K.T.) and a predoctoral fellowship from the Nakajima Foundation (to T.N.).

Competing interests

The authors declare no conflicts of interest or competing interests.

References

  1. Nobori, T., Velasquez, A. C., Wu, J., Kvitko, B. H., Kremer, J. M., Wang, Y., He, S. Y. and Tsuda, K. (2018). Transcriptome landscape of a bacterial pathogen under plant immunity. Proc Natl Acad Sci U S A 115(13): E3055-E3064.
  2. Anders, S., Pyl, P. T. and Huber, W. (2015). HTSeq–a Python framework to work with high-throughput sequencing data. Bioinformatics 31(2): 166-169.
  3. Langmead, B. and Salzberg, S. L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9(4): 357-35

简介

由于受感染植物组织中细菌RNA的丰度低,因此在植物中分析细菌转录组具有挑战性。 在这里,我们描述了一个描述叶子细菌病原体转录组的协议, Pseudomonas syringae pv。 番茄 DC3000,在感染早期的拟南芥叶中使用RNA测序(RNA-Seq)。 首先从感染的叶子中物理分离细菌细胞,然后进行RNA提取,植物rRNA消耗,cDNA文库合成和RNA-Seq。 该协议不仅适用于 A.拟南芥-P。 syringae 病理系统,但也适用于不同的植物 - 细菌组合。

【背景】植物已经进化出先天免疫系统以抵御病原体攻击。在过去的几十年中,已经深入研究了病原体识别和免疫信号传导途径的分子机制。然而,植物免疫如何影响病原体代谢以抑制病原体生长几乎不被理解,因为在植物中分析病原体反应很困难。在细菌病原体的情况下,植物叶内的转录组分析很难研究,因为细菌mRNA的量远低于植物的数量;由于植物中细菌的人口密度低,在感染的早期阶段尤其具有挑战性。为克服这一局限性,我们建立了一种从感染的植物叶片中分离细菌并用RNA-Seq分析细菌转录组的方法。该方法已成功用于分析模型细菌病原体 Pseudomonas syringae pv的转录组。 番茄 DC3000在模式植物 Arabidopsis thaliana 中的各种条件下(Nobori et al。,2018).

关键字:转录组测序技术, 细菌性病原体, 拟南芥, 转录组, 假单胞菌

材料和试剂

  1. 50毫升Falcon管(Corning,Falcon ®,目录号:352070)
  2. 1.5毫升Eppendorf Safe-Lock管(Eppendorf,目录号:0030120086)
  3. 移液器吸头,1,000μl和10μl体积(Corning,DeckWorks TM ,目录号:4124和4120)
  4. 无菌血清移液管,25 ml(Corning,目录号:4251)
  5. 1毫升无针注射器(BD,Luer-Lok TM ,目录号:303172)
  6. 4毫米不锈钢球(Mühlmeier)
  7. 6μm滤网(Bückmann,目录号:20000796)
  8. 30-32天龄拟南芥植物
  9. Pseudomonas syringae pv。 番茄 DC3000( Pto )
  10. 100%乙醇(VWR International,目录号:20821.321)
  11. 氯仿(Carl Roth,目录号:4432)
  12. peqGOLD TriFast(VWR,PeqLab,目录号:30-2010)
  13. 苯酚溶液pH 8.0(Sigma-Aldrich,目录号:P4557)
  14. 无核酸酶水(未经DEPC处理)(Thermo Fisher Scientific,Invitrogen TM ,Ambion ®,目录号:AM9932)
  15. 三(2-羧乙基)膦(TCEP)(Sigma-Aldrich,目录号:C4706)
  16. RNAqueous ® -Micro Total RNA Isolation Kit(Thermo Fisher Scientific,Invitrogen TM ,Ambion ®,目录号:AM1931)
  17. TURBO无DNA试剂盒(Thermo Fisher Scientific,Invitrogen TM ,Ambion ®,目录号:AM1907)
  18. Ribo-Zero rRNA去除试剂盒(植物)(Illumina,目录号:MRZPL1224)
  19. RNeasy Mini Kit(QIAGEN,目录号:74104)
  20. Ovation ®完整的原核RNA-Seq DR多重系统(NuGEN Technologies,目录号:0326-32)
  21. QIAGEN-tip 500(QIAGEN,目录号:10063)
  22. 液氮
  23. NaOH(Carl Roth,目录号:6771)
  24. 细菌分离缓冲液(见食谱)

设备

  1. Eppendorf参考移液器(Eppendorf,100-1,000μl)
  2. Gilson PIPETMAN经典移液器(Gilson,型号:P20,目录号:F123600)
  3. 电动移液器(BrandTech Scientific,型号:accu-jet ® pro,目录号:26330)
  4. 冷冻离心机(Eppendorf,型号:5417 R和5810 R)
  5. 辊式搅拌机SRT1(Stuart Scientific,型号:SRT1)
  6. 涡旋混合器(科学工业,型号:Vort ex-Genie 2,目录号:SI-0236)
  7. 振动筛
  8. 热循环仪(DNA Engine Tetrad 2 Peltier热循环仪,Bio-Rad,型号:Tetrad 2)
  9. NanoDrop TM One / One C 微量体积紫外 - 可见分光光度计(Thermo Fisher Scientific,Thermo Scientific TM ,型号:NanoDrop TM One C ,目录号:ND-ONE-W)
  10. -80°C冰箱
  11. 高压灭菌器
  12. 通风柜
  13. 镊子(精细科学工具,型号:Dumont#5,目录号:11251-10)
  14. 塑料刀具
  15. 剪刀
  16. Illumina HiSeq-3000测序仪

软件

  1. BowTie2
  2. Python包HTSeq

程序

程序概述如图1所示。


图1.植物细菌转录组分析的工作流程摘要。该图采用了Nobori 等(2018), PNAS 。叶子感染了 P.将丁香(syringae)粉碎并在细菌分离缓冲液中温育,然后过滤掉大的植物组织。然后将流过的血液离心以将细菌细胞与植物细胞分离。收获细菌细胞并进行RNA提取,cDNA文库制备和RNA-seq。


  1. 植物生长状况
    使植物在22℃的室中生长10小时光照期和60%相对湿度24天,然后在另一个室中在22℃生长12小时光照期和60%相对湿度。光强度为约120μmol/ m 2, 2 / sec。三十到三十二天 A。使用拟南芥植物。

  2. 细菌分离(第1天)
    注意:接触冷冻管时请使用防护设备。
    1. 将细菌悬浮液(OD 600 = 0.5)浸入 A.从远轴侧用1ml无针注射器离开(4片叶/植物×20株植物,总共80片,约3g)拟南芥叶片。确保整个叶子区域渗入细菌。
    2. 浸润6小时后,用镊子将80片叶子收集到含有8-10个不锈钢球(4毫米;用乙醇洗涤并风干)中的50毫升Falcon管中。用液氮快速冷冻。样品可以在-80°C下储存。
    3. 用手彻底摇匀,将叶子压成小块(图2)。确保样品在压碎过程中不会解冻。


      图2.在细菌分离缓冲液中孵育之前的碎叶样品

    4. 在通风橱中加入30ml冰冷的细菌分离缓冲液,立即涡旋10秒,然后用手剧烈摇动10秒。使用辊式混合器SRT1在4°C下以33rpm振荡孵育管20小时。

  3. 细菌分离(第2天)
    注意:步骤C1-C7和D1-D12应在通风橱中完成。 
    1. 装备50毫升猎鹰管,过滤器支架(由QIAGEN-tip 500柱手工制作;上半部分用塑料切割器切割;使用前高压灭菌)和单层6微米过滤网(切成12厘米x 15)厘米大小,然后高压灭菌)(图3)。用剪刀修剪多余的过滤网,使盖子适合管子。


      图3.配备50 ml Falcon管的过滤器和过滤器支架。 A.切割QIAGEN-tip 500色谱柱,制成过滤器支架。 B. 50毫升Falcon管,6微米滤网(12厘米×15厘米)和过滤器支架。 C.将50毫升Falcon管装上过滤器支架和滤网。 D.修剪掉多余的过滤网,使盖子能够与管子配合。 

    2. 将样品(15毫升;不含不锈钢球)施加到柱上,并在4℃下通过以1,300 x g 离心10秒(或直到所有液体通过)过滤它。在保持管内流通的同时,将剩余的样品(15 ml)应用于色谱柱并重复过滤。
    3. 取下过滤器和过滤器支架。在4℃下以3,200 x g 离心流过20分钟。用装有25ml血清移液管的电子移液管丢弃上清液。您可以留下一些液体(100μl)以避免样品丢失。
    4. 加入900μl冰冷的细菌分离缓冲液并通过涡旋重悬沉淀,然后通过移液将其转移到冰上新的1.5ml管中。确保颗粒完全重悬。
    5. 在4℃以2,300 x g 离心20分钟。您将看到两层颗粒:顶部为白色,底部为绿色(图4)。
      注意:有时管的上部变成淡绿色。在这种情况下,您可以通过用少量液相移液去除绿色部分并丢弃它以避免潜在的植物污染。


      图4.双层颗粒由植物(底部,绿色)和细菌(顶部,白色)组成

    6. 装备20μlGilson移液器和10μl滤嘴(容量为20μl),并将体积设置为18μl。通过靠近颗粒表面上下移液,小心地仅重悬顶部白色层。当缓冲液被细菌细胞混浊时,使用1,000μl移液管将缓冲液(约1,000μl)转移到冰上新的1.5 ml管中。然后,小心地加入1毫升新的冰冷细菌分离缓冲液,不要通过平铺管破坏沉淀物。重复重新悬浮并在新的1.5 ml管中收集细菌细胞悬浮液(总共三次或直到白色层完全去除)。
      注意:
      1. 您可以使用适合移液器的较大吸头,但较小的吸头可以更容易地重悬细菌层而不会破坏植物层。
      2. 如果植物组织层塌陷并与细菌细胞层混合,您可以通过涡旋完全重悬浮整个沉淀然后重复离心步骤(步骤C5)来恢复细菌层。
    7. 将细菌悬浮液在10,000 x g 下离心2分钟以收获细菌细胞。通过移液器丢弃上清液。

  4. RNA提取
    1. 每粒样品加入1毫升peqGOLD TriFast颗粒。例如,如果在步骤B6之后有3个试管,则向每个试管中加入333μlpeqGOLDTriFast,涡旋,并将它们合并在一个试管中。样品可以在-80°C下储存。
    2. 每1ml peqGOLD TriFast加入200μl氯仿,涡旋,并在室温(RT)下离开管2分钟。
    3. 在4℃以12,000 x g 离心15分钟。通过移液将含水部分(通常400-500μl)加入新的1.5ml Eppendorf管中。
    4. 根据制造商的方案,使用RNAqueous ® -Micro总RNA分离试剂盒(Invitrogen,Ambion ®)分离RNA,并进行一些修改(见下文)。
    5. 将洗脱液预热至75°C。
    6. 向样品中加入半体积的100%乙醇并短暂涡旋。
    7. 将样品(最多150μl)加载到Micro Filter Cartridge组件上。在室温下以12,000 x g 离心10秒。重复此步骤,直到加载所有样品。丢弃流通。
    8. 将180μl洗涤液1加入Micro Filter Cartridge中。在室温下以12,000 x g 离心10秒。
    9. 将180μl洗涤液2/3加入Micro Filter Cartridge中。在室温下以12,000 x g 离心10秒钟。
    10. 再一次重复步骤C9。
    11. 将微滤盒更换为新的收集管。在室温下以12,000 x g 离心1.5分钟以除去残留的流体并干燥过滤器。
    12. 将Micro Filter Cartridge更换为新的1.5 ml Eppendorf管。用15μl预热的洗脱液洗脱RNA。再次将洗脱重复洗涤到同一管中(总共30μl)。
    13. 根据制造商的方案,使用TURBO无DNA试剂盒(Ambion ®)进行DNA酶处理。
    14. 使用NanoDrop检查RNA浓度。典型的RNA浓度为100-200 ng /μl。

  5. 植物来源的rRNA耗尽
    遵循Ribo-Zero rRNA去除试剂盒(植物)的方案。如制造商的方案中所述,使用RNeasy Mini Kit(QIAGEN)进行RNA纯化。典型的RNA产量为100-400ng。
  6. 图书馆准备和排序
    1. 使用Ovation ®完全原核RNA-Seq DR多重系统(NuGEN)根据制造商的方案合成cDNA文库,并进行一些修改。
      重要提示:输入10 ng富含细菌的RNA,尽管该试剂盒推荐使用100-500 ng。这是因为你不能总是获得这种丰富的RNA。保持RNA输入量一致。
    2. 使用Illumina HiSeq测序仪对文库进行测序。

数据分析

转录组的Illumina读数的定位和计数:

  1. 序列读数映射在 Pto 基因组上(可从 http://pseudomonas.com/ 获得)使用BowTie2(Langmead和Salzberg,2012)。将phred质量分数大于33的序列读数作为输入(命令:--phred33)。我们对其他参数使用默认设置。
  2. 使用Python软件包HTSeq(Anders et al。,2015)使用默认设置计算映射读数。 Pto 的基因注释文件(GFF3文件)可在 http://pseudomonas.com/

笔记

  1. 苯酚和氯仿都是有毒的。将它们放在通风橱中并穿戴适当的保护装置。
  2. 在步骤D14中,RNA的质量趋于变化且通常较低。然而,在我们之前的实验中,这并没有引起数据重现性和RNA-Seq测量准确性的问题(Nobori et al。,2018)。

食谱

  1. 细菌分离缓冲液
    无核酸酶水
    25mM TCEP(三(2-羧乙基)膦),pH 4.5用10N NaOH调节
    9.5%乙醇
    0.5%苯酚溶液
    注意:每次都准备好它。

致谢

该协议改编自先前发表的论文(Nobori et al。,2018)。这项工作得到了Max Planck Society和Deutsche Forschungsgemeinschaft Grant SFB670(到K.T.)以及Nakajima Foundation(到T.N.)的博士后奖学金的支持。

利益争夺

作者声明没有利益冲突或竞争利益。

参考

  1. 1.Nobori,T.,Velasquez,A.C.,Wu,J.,Kvitko,B.H.,Kremer,J.M.,Wang,Y.,He,S.Y。和Tsuda,K。(2018)。 植物免疫下细菌病原体的转录组景观。 Proc Natl Acad Sci美国 115(13):E3055-E3064。
  2. Anders,S.,Pyl,P。T.和Huber,W。(2015)。 HTSeq-用于处理高通量测序数据的Python框架。 生物信息学 31(2):166-169。
  3. Langmead,B。和Salzberg,S。L.(2012)。 与Bowtie 2进行快速读取对齐。 Nat方法 9(4):357-35
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引用:Nobori, T. and Tsuda, K. (2018). In planta Transcriptome Analysis of Pseudomonas syringae. Bio-protocol 8(17): e2987. DOI: 10.21769/BioProtoc.2987.
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