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Sep 2017

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Low-cost and Multiplexable Whole mRNA-Seq Library Preparation Method with Oligo-dT Magnetic Beads for Illumina Sequencing Platforms
Oligo-dT磁珠用于Illumina测序平台的低成本和可复用的完整mRNA-Seq文库制备方法   

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Abstract

RNA-Seq is a powerful method for transcriptome analysis used in varied field of biology. Although several commercial products and hand-made protocols enable us to prepare RNA-Seq library from total RNA, their cost are still expensive. Here, we established a low-cost and multiplexable whole mRNA-Seq library preparation method for illumine sequencers. In order to reduce cost, we used cost-effective and robust commercial regents with small reaction volumes. This method is a whole mRNA-Seq, which can be applied even to non-model organisms lacking the transcriptome references. In addition, we designed large number of 3′ PCR primer including 8 nucleotides barcode sequences for multiplexing up to three hundreds samples. To summarize, it is possible with this protocol to prepare 96 directional RNA-Seq libraries from purified total RNA in three days and can be pooled for up to three hundred libraries. This is beneficial for large scale transcriptome analysis in many fields of animals and plant biology.

Keywords: RNA-Seq (RNA-Seq), mRNA (mRNA), Multiplexing (复用), Transcriptome (转录组), Illumina (Illumina平台)

Background

In the last decade, sequencing cost has been reduced drastically thanks to the advance in massive parallel sequencing technologies (Muir et al., 2016). On the other hand, the cost at library preparation step stands out. For example, commercial kits for RNA-Seq library preparation such as TruSeq RNA Library Prep Kit (illumine, USA) and NEBNext Ultra RNA Library Prep Kit (NEB, USA) cost around 40-60 dollars per sample. Recently, several studies have developed cost-effective RNA-Seq library preparation methods (Kumar et al., 2012; Nagano et al., 2015; Townsley et al., 2015; Alpern et al., 2019; Kamitani et al., 2019). Particularly, 3′ RNA-Seq protocols (Lasy-Seq and BRB-Seq), enable early-pooling of samples resulting in reducing the cost into about two dollar per sample (Alpern et al., 2019; Kamitani et al., 2019). While 3′ RNA-Seq is economically superior to whole mRNA-Seq method, whole mRNA-Seq can sequence full-length of RNA. Thus, whole mRNA-Seq is good for detection of splicing variants and novel transcripts. Although our previously-developed protocol of whole mRNA-Seq using rRNA depletion is cheap (Nagano et al., 2015), it requires many kinds of antisense oligo against rRNA and enzymes for reverse transcription, 2nd strand synthesis, end-repair, A-tailing, adapter ligation and library amplification. In this protocol, (1) we replaced rRNA depletion against mRNA-purification using oligo-dT beads in order to reduce initial cost. (2) Enzymes required for end-repair to library amplification were replaced with KAPA Hyper Prep Kit (Roche, Switzerland); this simplification increases reaction efficiency as well as reduce labor. (3) We saved reaction volumes throughout all steps (≤ 20 µl), which saves cost and enables handling even with 384-well plates. The required cost is approximately one-third of the golden standard kits. (4) We designed 300 kinds of 3′ PCR primers with barcode sequences for multiplexing, which enabled us to sequence 300 samples at a time with a cost-effective and ultra-high-throughput sequencer such as NovaSeq.

Materials and Reagents

  1. 0.2 ml PCR tubes
  2. Pipette tips
  3. 5× Super Script IV buffer (Invitrogen)
  4. DTT (Invitrogen)
  5. Dynabeads® Oligo (dT)25 (Thermo Fisher scientific)
  6. Total RNA
  7. Random primer (N)6 (TaKaRa)
  8. dNTP (25 mM each) (Promega)
  9. SuperScript IV (Invitrogen)
  10. Actinomycin D (1,000 ng/μl) (Nacalai Tesque)
  11. AMPure XP (Beckman Coulter)
  12. EtOH
  13. Nuclease-free water
  14. 10× Blue Buffer (Enzymatics)
  15. RNase H (Enzymatics)
  16. DNA polymerase I (Enzymatics)
  17. KAPA Hyper prep kit (KAPA Biosystems)
  18. Tris-HCl
  19. LiCl
  20. EDTA
  21. DNA glycosylase (UDG) (Enzymatics)
  22. KAPA HiFi HotStart ReadyMix (2×) (KAPA Biosystems)
  23. KAPA Library Quantification Kit Illumina (KAPA Biosystems)
  24. Agilent High Sensitivity DNA kit (Agilent Technologies)
  25. 2× binding buffer (see Recipes)
  26. Washing buffer (see Recipes)

Equipment

  1. MagnaStand YS-model (FastGene)
  2. Agilent 2100 Bioanalyzer (Agilent Technologies)
  3. Quantitative PCR instrument
  4. Pipette
  5. Thermal cycler

Procedure

In this protocol, we used MagnaStand YS-model (FastGene) in nucleotide acid purification with magnetic beads. All experiments were conducted in 0.2 ml PCR tubes. Reaction solutions should be mixed by tapping or inversion. All operations should be conducted under a DNA/RNA-free environment. The overview of this protocol is shown in Figure 1.


Figure 1. The protocol flow chart of this protocol. The approximate time required is shown for each step.

  1. mRNA purification
    1. Wash 15 μl of Dynabeads® Oligo (dT)25 (Thermo Fisher Scientific) twice each with 50 μl of 2× binding buffer (Recipe 1) using Magna Stand for 0.2 ml PCR Tube (FastGene) and then resuspend the beads in 30 μl of 2× binding buffer for the later use.
      ‘Wash’ indicates:
      1. Put on a magnet, 5 min, remove supernatant.
      2. Add 2× binding buffer and mix by pipetting.
      3. Repeat.
    2. Denature total RNA (more than 1 μg and RIN > 7.0 is recommended) in 30 μl of distilled water or 10 mM Tris·HCl (pH 8.5) at 65 °C for 2 min and then immediately transfer onto ice. All thermal control procedures in this protocol can be processed in a thermal cycler. Then add 30 μl of the washed Dynabeads® Oligo (dT)25. Mix the mixture well by pipetting and incubate at room temperature for 10 min. And next wash the mixture with 70 μl of washing buffer (Recipe 2) twice using the Magna Stand.
      ‘Wash’ indicates:
      1. Put on a magnet, 5 min, remove supernatant.
      2. Add washing buffer and mix by pipetting.
      3. Repeat.
    3. Elute the RNA in 30 μl of pre-warmed distilled water at 80 °C for 5 min and then immediately chill on ice. For a second round of polyA-selection, add 30 μl of 2× binding buffer to the solution, and incubate at room temperature for 10 min. Again, wash the mixture with 70 μl of washing buffer twice using the Magna Stand. Elute the RNA in 14 μl of pre-warmed distilled water at 80 °C for 2 min, then immediately place on the magnet and collect into a new tube. The expected volume of the product is 13 μl, with which the following experiments may only be performed twice at most.

  2. RNA-seq library preparation and sequencing
    1. Mix 5 μl of purified mRNA obtained in the above section with 4 μl of 5× Super Script IV buffer (Invitrogen) and 1 μl of frozen stock of 100 mM DTT (Invitrogen).
    2. mRNA is fragmented by incubating at 94 °C for 4.5 min and then immediately cooled down on ice.
    3. Then add 0.6 μl of 100 μM random primer (N)6 (TaKaRa) and 0.9 μl of distilled water to the fragmented mRNA. The volume of the mix is 12 μl.
    4. Incubate the mixture at 50 °C for 5 min and immediately chill on ice to relax the secondary structures of the mRNA.
    5. Add the reverse transcription master mix (1 μl of frozen stock of 100 mM DTT, 0.4 μl of dNTP (25 mM each) (Promega); 0.1 μl of SuperScript IV (Invitrogen); 0.2 μl of Actinomycin D (1,000 ng/μl) (Nacalai Tesque); and 5.9 μl of distilled water). Now the volume of the mix is 20 μl.
    6. For the reverse transcription step, incubate the mixture at 25 °C for 10 min, followed by 10 min at 50 °C. Inactivate SuperScript IV by heating the mixture at 80 °C for 15 min. Then add 24 μl of AMPure XP (Beckman Coulter) and 12 μl of 99.5% EtOH, and perform the purification step according to the manufacturer’s manual.
      The detail of purification with AMPure XP beads in this protocol is as below:
      1. Mix by pipetting.
      2. Room temperature, 5 min.
      3. Put on magnet, 5 min, remove supernatant.
      4. Add 70 µl of 70% EtOH on the magnet stand and remove it. Repeat this step again.
      5. Dry up for 1 min.
      6. Add 10.0 µl or other Nuclease free water, mix by pipetting.
      7. R.T. for 1 min.
      8. Put on magnet, collect 10.0 µl or other of supernatant to 384-well PCR plate.
    7. Elute the transcription product with 10 μl of distilled water. Mix the purified DNA/RNA hybrid solution without beads with the second strand synthesis master mix [2 μl of 10× Blue Buffer (Enzymatics), 1 μl of dUTP/NTP mix (Fermentas), 0.5 μl of frozen stock of 100 mM DTT, 0.5 μl of RNase H (Enzymatics), 1 μl of DNA polymerase I (Enzymatics), and 5 μl of distilled water] The final volume of the mixture is 20 μl.
    8. Incubate the mixture at 16 °C for 4 h. Purify dsDNA with 24 μl of AMPure XP according to the manufacturer’s manual. Elute the purified dsDNA with 10 μl of distilled water. Use 5 μl of the dsDNA solution for the following step.
    9. Perform end-repair, A-tailing and adapter ligation using a KAPA Hyper Prep kit (KAPA Biosystems) with 1/10× volume of the solutions according to the manufacturer’s manual. Use 1 μl of 0.1 μM Y-shape adapter (Recipe 3, Nagano et al., 2015) in the adapter ligation step and incubate for 15 min at 20 °C. The final volume of this reaction product is 11 μl.
    10. Perform size selection of the ligation product with 5.5 μl of AMPure XP, resulting in 0.32× AMPure XP. The first size-selection is conducted at the smaller ratio than the second due to strong viscosity of ligation reaction mix. Elute the purified dsDNA using 10 μl of distilled water. Carry out the second round of size selection with 10 μl of AMPure XP, resulting in 0.5× AMPure XP. Elute the size-selected ligation product with 15 μl of 10 mM Tris-HCl, pH 8.0. Add 1 μl of uracil DNA glycosylase (UDG) (Enzymatics) to the size-selected ligation product. The volume of the mix is 16 μl.
    11. Incubate the mixture at 37 °C for 30 min to exclude the second-strand DNA.
    12. For library amplification, mix 2 μl of the UDG-digested DNA with 1 μl of 2.5 μM index primer (CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGT, XXXXXXXX indicates index sequence in the supplemental file) (Nagano et al., 2015), 1 μl of 10 μM universal primer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) (Nagano et al., 2015), 0.5 μl of distilled water and 5 μl of KAPA HiFi HotStart ReadyMix (2×) (KAPA Biosystems). The volume of the mix is 10 μl.
    13. Amplify DNA fragments with the adapters and an index sequence using a thermal cycler with the following program: denature at 94 °C for 2 min, 18 cycles at 98 °C for 10 s, 65 °C for 30 s, 72 °C for 30 s as an amplification step, and 72 °C for 5 min for the final extension. Then perform two rounds of size selection to remove adapter dimer with an equal volume of AMPure XP to the library solution with 10 µl of D.W. for elution. Next elute the purified library with 10 μl of distilled water. Now, you can pool the libraries, if needed. Also use library quantification kit (e.g., KAPA Library Quantification Kit Illumina (KAPA Biosystems)) to determine the concentration followed by pooling.
    14. Take out 1 μl of the purified library for electrophoresis using an Agilent High Sensitivity DNA kit (Agilent Technologies) to evaluate quality. The typical concentration of the library is more than 1 ng/µl, and the size distribution is shown below (Figure 2).


      Figure 2. The typical size distribution of the final product of this protocol. A result from electrophoresis produced with Agilent High Sensitivity DNA kit.

Recipes

  1. 2× binding buffer
    40 mM Tris-HCl, pH 7.6
    2 M LiCl
    4 mM EDTA
  2. Washing buffer
    10 mM Tris-HCl, pH 7.6
    0.15 M LiCl
    1 mM EDTA
  3. Y-shape adapter
    A mixture of 100 mM adapters (5′-A*\A*TGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT*C*T-3′ 5′-/5Phos/-G*A*TCGGAAGAGCACACGTCTGAACTCCAGTC*A*C-3′.
    *signifies a phosphonothioate bond. /5Phos/signifies a phosphorylation) is annealed using a thermal cycler with the following program:
    95 °C for 2 min, slow-cooled to 25 °C (0.1 °C/s), followed by 30 min at 25 °C. The annealed adapter (50 μM) is diluted into 0.1 μM with D.W and stored at -20 °C.

Acknowledgments

This work was supported by the JST CREST JPMJCR15O2 to A.J.N. This protocol was modified from the method described in Nagano et al., 2015.

Competing interests

We have no conflicts of interest or competing interests.

References

  1. Alpern, D., Gardeux, V., Russeil, J., Mangeat, B., Meireles-Filho, A. C. A., Breysse, R., Hacker, D. and Deplancke, B. (2019). BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing. Genome Biol 20(1): 71.
  2. Kamitani, M., Kashima, M., Tezuka, A. and Nagano, A. J. (2019). Lasy-Seq: a high-throughput library preparation method for RNA-Seq and its application in the analysis of plant responses to fluctuating temperatures. Sci Rep 9(1): 7091.
  3. Kumar, R., Ichihashi, Y., Kimura, S., Chitwood, D. H., Headland, L. R., Peng, J., Maloof, J. N. and Sinha, N. R. (2012). A high-throughput method for Illumina RNA-Seq library preparation. Front Plant Sci 3: 202.
  4. Muir, P., Li, S., Lou, S., Wang, D., Spakowicz, D. J., Salichos, L., Zhang, J., Weinstock, G. M., Isaacs, F., Rozowsky, J. and Gerstein, M. (2016). The real cost of sequencing: scaling computation to keep pace with data generation. Genome Biol 17: 53.
  5. Nagano, A. J., Honjo, M. N., Mihara, M., Sato, M. and Kudoh, H. (2015). Detection of plant viruses in natural environments by using RNA-Seq. Methods Mol Biol 1236: 89-98. 
  6. Townsley, B. T., Covington, M. F., Ichihashi, Y., Zumstein, K. and Sinha, N. R. (2015). BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction. Front Plant Sci 6: 366.

简介

[摘要 ] RNA的序列是转录组分析使用的有效方法的变种d 灭蝇灯领域的生物。尽管一些商业产品和手工制作的协议使我们准备RNA的序列从总RNA库,其成本仍然是昂贵的。 ,我们建立了低成本和复用的全mRNA- SEQ 用于照亮sequencers.In顺序文库制备方法以降低成本,我们使用成本有效的具有小反应volumes.This方法和强大的商业试剂是一个整体mRNA- SEQ ,这甚至可以应用于非模式生物缺乏转录引用。在另外,我们设计的大号码3 ' PCR引物,包括8个核苷酸条码序列复用多达三个数百个样品,为了总表我ž E,有可能用这协议,以制备96个定向RNA- SEQ 从纯化的总RNA文库在三天,并且可以合并为至多三百libraries.This是许多FIE大规模转录组分析有益 动物和植物生物学研究。

[背景 ] 在过去的十年,测序成本已经drasti减少卡利得益于一个在大规模并行测序技术dvance (缪尔等人。,2016) 。对另一方面,在文库制备步骤的成本代表out.For EXA mple ,商业试剂盒对RNA的序列库制剂如TruSeq RNA文库制备试剂盒(照亮,美国)和NEBNext 超RNA文库制备试剂盒(NEB,USA)的成本大约40 - 60美元,每样最近,一些研究已经开发成本- RNA-有效SEQ 文库制备方法(库马尔等人,2012 ;.长野等人,2015 ;.汤斯利等人,2015 ;. 阿尔珀恩等人,2019 ;.上谷等人。,2019) 。特别是,3 ' RNA- SEQ 协议(镭石光电-SEQ 和BRB- SEQ ),使得能够早期汇集,导致降低了成本为每样品约两美元样品(阿尔珀恩等人,2019;上谷等人。,2019) 。而3 ' RNA- Seq 在经济上优于完整的mRNA- Seq 方法,完整的mRNA- Seq 可以测序全长RNA,因此,完整的mRNA- Se Q 是良好的检测剪接变异体和新颖的成绩单。虽然我们以前开发协议全MRNA- SEQ 使用rRNA的消耗很便宜(长野等人,2015年),它需要许多种反义益反对rRNA基因和酶有关反向转录,第二链合成,末端修复,A尾修饰,衔接子连接和文库扩增。在此方案中,(1 )我们使用寡聚dT 珠替换了rRNA 消耗对mRNA的纯化,以降低初始成本。(2 )酶所需的F 或Ë ND-修复文库扩增分别替换d 与KAPA的Hyper制备试剂盒(Roche,瑞士);这简化增加反应效率以及降低劳动。(3 )我们在所有步骤保存的反应体积(≤ 20微升),从而节省了成本,甚至与384孔plates.The需要的成本使得能够处理是大约三分之一的黄金标准试剂盒。(4 )我们设计300种3 ' PCR条码序列的引物,用于多路复用,WH ich使我们能够使用具有成本效益的超高通量测序仪(例如NovaSeq)一次对300个样品进行测序。

关键字:RNA-Seq, mRNA, 复用, 转录组, Illumina平台

材料和试剂


 


1. 0.2 ml PCR管      


2. 移液器技巧      


3. 5 × Super Script IV缓冲区(Invitrogen)      


4. DTT(Invitrogen)      


5. 磁珠® 的Oligo(DT )25(热Fisher Scientific公司)      


6. 总RNA      


7. 随机引物(N)6 (TaKaRa )。      


8. DNTP(每个25 MM )(Promega )      


9. SuperScript IV(Invitrogen)      


10. 放线菌素D(1,000 Ng / Myu L )(Nacalai Tesque )   


11. AMPure XP(贝克曼库尔特)   


12. 乙醇   


13. 核酸酶- 自由水   


14. 10×蓝色缓冲液(酶)   


15. R Nase H(酶)   


16. DNA聚合酶I(酶)   


17. KAPA Hyper准备套件(KAPA Biosystems)   


18. Tris-HCl   


19. 氯化锂   


20. EDTA   


21. DNA糖基化酶(UDG)(酶)   


22. KAPA HiFi HotStart ReadyMix (2×)(KAPA Biosystems)   


23. KAPA库定量试剂盒Illumina(KAPA Biosystems)   


24. 安捷伦高灵敏度DNA试剂盒(安捷伦科技公司)   


25.2 ×绑定缓冲区(请参阅食谱)   


26. 洗涤缓冲液(请参阅食谱)   


 


设备


 


MagnaStand YS模型(FastGene )
Agilent 2100 生物分析仪(安捷伦科技公司)
定量PCR仪
吸管
热循环仪
 


程序


 


在这个协议中,我们使用MagnaStand YS-模型(FastGen ê 在核苷酸酸纯化)与磁性beads.All 实验在0.2ml PCR管中进行。反应方案应该是混合通过轻敲或inversion.All 操作小号应该下一个进行无DNA / RNA的环境。该协议的概述如图1所示。


 


D:\ Reformatting \ 2020-4-7 \ 1902970--1419鹿岛诚554819 \ Figs jpg \ Fig 1.jpg


1图。该协议流程图此协议。在一个Pproximate 时间要求,显示了每个步骤。


 


mRNA纯化
洗15 MYU 大号中的Dynabeads ® 寡(DT )25(热费舍尔小号系统求解)两次,每次用50 Myu L Of 2×结合缓冲液 (方案1)使用Magna Stand制备0.2 Ml PCR管(FastGene ),然后将微珠重悬于30 Myu L 的2×结合缓冲液中,以备后用。
“洗涤”表示:


穿上一个磁铁,5分钟,收集除去。
加入2×结合缓冲液并通过移液混合。
再说一次。
Ť变性Otal RNA(超过1 Myug 和RIN> 7.0建议)在30ml MYU 大号的蒸馏水或者10米MM 的Tris·HCl的(PH 8.5)在65℃下2分钟,并 然后立即转移Ø ñ 为了冰。一个的L1 热控程序在这个协议可以处理在热循环仪。然后加入30 MYU 大号对洗过的磁珠® 的Oligo(DT )25 。混合牛逼他混好了,通过移液和孵化在ř OOM温度10分钟。 而接下来洗牛逼他混合物70 MYU 大号中洗涤缓冲液(配方2)两次使用的麦格纳立场。
“洗涤”表示:


穿上一个磁铁,5分钟,收集除去。
加入洗涤缓冲液并通过移液混合。
再说一次。
Ť洗脱他RNA在30 MYU 大号预温热蒸馏水80℃5分钟,并然后立即冰上冷却。对于第二轮的PolyA -Selection,添加30 MYU 大号中2 × 结合缓冲液至溶液中,然后在室温下孵育10分钟,再用Magna Stand 用70 Myu L 的洗涤缓冲液两次洗涤混合物,在80°C的14 Myu L 预热蒸馏水中洗脱RNA 2分钟,然后的Plac立即Ë 上的磁体和收集在新的管。该预期量的产品是13 MYU 大号,与在下面的实验可能只进行两次最多。
 


RNA- seq 文库的制备和测序
混合5 MYU 大号纯化mRNA获得在上面的部分具有4 MYU 大号5×超级脚本IV缓冲液(Invitrogen)和1 MYU 大号冷冻库存100 MM DTT(Invitrogen)中。
的mRNA 是通过分段孵育在94℃进行4.5分钟,然后立即凉编在冰上。
然后添加0.6 MYU 大号100 MyuM 随机引物(N )6 (TaKaRa公司)和0.9 MYU 大号蒸馏水要的碎片MRNA 。卷的混合物12 MYU 大号。
将其在50°C下孵育5分钟,然后立即在冰上冷却以放松M RNA 的二级结构。
添加逆转录主混合物(1 亩升100的冷冻原液毫DTT,0.4 亩升的dNTPs(25 毫摩尔的每个)(Promega公司),0.1 亩升的的SuperScript IV(Invitrogen公司),0.2 亩升的放线菌素d (千NG / MYU 大号)(ナTesque公司)和5.9 MYU 大号蒸馏的水)。现在牛逼他卷的搭配是20 MYU 大号。    
用于逆转录步骤,孵育该混合物在25℃下10分钟,接着用10分钟,在50℃下失活的SuperScript IV通过加热将混合物在80℃下15分钟。然后加入24 MYU 大号中AMPure XP( Coulter Beckman)和99.5 Pasento EtOH的12 Myu L ,并根据制造商手册执行纯化步骤。
该协议中使用AMPure XP珠子进行纯化的细节如下:


通过移液混合。
室温5分钟。
戴上磁铁5分钟,除去总量。
在磁座上加入70 µl 70%的乙醇并取出。再次重复此步骤。
干涸为1分钟。
加入10.0 µl或其他无核仁水,通过移液混合。
室温1分钟。
穿上磁铁,收集10.0微升或其他上清液384 - 孔PCR板。
洗脱Ť 他转录产物用10 MYU 大号蒸馏水。中混合Ť 他纯化的DNA / RNA杂交解决方案,而珠随着该第二链合成主混合物[ 2 MYU 大号10×蓝色缓冲液(Enzymatics ),1 MYU 大号中的dUTP / NTP混合物(Fermentas公司),0.5 亩升100的冷冻原液毫DTT,0.5 亩升RNA酶H(的Enzymatics ),1 亩升DNA聚合酶I(Enzymatics )和5 微米升蒸馏水] 的最终的体积混合真实姿态是20 MYU 大号。
孵育Ť 他混合物在16℃下对于4 H. 净化的dsDNA 随着24 MYU 大号中AMPure XP根据制造商的手册。洗脱Ť 他纯化双链DNA用10 MYU 大号蒸馏水。使用5 MYU 大号的双链DNA解为接下来的步骤。
根据制造商手册,使用KAPA Hyper P Rep试剂盒(KAPA Biosystems)以1/10×体积的溶液进行E Nd修复,A尾部和适配器连接,使用1 Myu L 的0.1 Myu M Y型适配器(配方3,长野等人。,2015)在适配器连接步骤和温育15分钟,阿Ť 20 ℃下。最终体积该反应产物是11 MYU 大号。
执行小号我泽选择结扎产品采用5.5 Myueru 中AMPure XP,导致0.32 × AMPure XP。第一大小选择中进行比第二受强粘性连接反应混合液。较小的比例洗脱牛逼他纯化使用10双链DNA MYU 大号的蒸馏水。贯彻牛逼,他第二轮尺寸选择用10 MYU 大号中AMPure XP,导致0.5 × AMPure XP。洗脱牛逼他尺寸选择结扎产品采用15 MYU 大号10 MM 三-的HCl ,PH 8.0。添加1 MYU 大号尿嘧啶DNA糖基化酶(UDG)(Enzymatics )的大小,选定的连接产物。的混合的体积为16 MYU 大号。
将混合物在37°C下孵育30分钟,以排除第二链DNA。
用于文库扩增,中号九2 MYU 大号的UDG消化的DNA 用1 MYU 大号2.5 MYU 中号索引引物(ShieieijishieijieieijieishijijishieitieishijieijieitiEkkusuEkkusuEkkusuEkkusuXXXXGTGACTGGAGTTCAGACGTGT,XXXXXXXX表示索引序列中的补充文件)(长野等人,2015) ,1 MYU 大号中10 MYU 中号通用引物(EieitijieitieishijijishijieishishieishishijieijieitishitieishieishitishititiTCCCTACACGACGCTCTTCCGATCT) (长野等人。,2015),0.5 MYU 大号蒸馏水和5 MYU 大号中KAPA 高保真的HotStart 预拌(2×)(KAPA Biosystems)中。该卷的混合物10 MYU 大号。
AMPLIFY DNA片段与所述适配器和使用热循环仪具有以下程序索引序列:变性94℃2分钟,在98℃下18个循环持续10秒,65℃30秒,72℃30 S作为扩增步骤,和72℃5分钟的最后延伸。然后执行Ť 禾轮尺寸选择要删除适配器二聚体的等体积的AMPure XP到库解具Myueru 10 DW进行洗脱。接着牛逼洗脱他纯净库10 MYU 大号蒸馏水。现在,你可以游泳池的图书馆,如果需要的话。也可以使用图书馆定量试剂盒(ē .G 。,KAPA文库定量试剂盒Illumina公司(KAPA生物系统公司))确定浓缩后通过合并。
拿出1 MYU 大号的纯化图书馆供选Rophoresis使用安捷伦高灵敏度DNA试剂盒(Agilent技术)来评估质量。典型的浓度库超过1纳克/ MYU L,并且大小分布如下所示(图URE 2)。
 


D:\ Reformatting \ 2020-4-7 \ 1902970--1419鹿岛诚554819 \ Figs jpg \ Fig2.jpg


2图。该方案最终产品的典型尺寸分布,这是使用安捷伦高灵敏度DNA试剂盒进行电泳得到的结果。


 


菜谱


 


2 x结合缓冲液
40 mM的Tris-HCl ,pH 7.6


2 M 氯化锂


4 毫米EDTA


洗涤缓冲液
10 mM的Tris-HCl ,pH 7.6


0.15 M 氯化锂


1 毫米EDTA


Y型适配器
100 mM 适配器的混合物(5'-A * \ A * TGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT * C * T-3'5'-/ 5Phos / -G * A * TCGGAAGAGCACGTCTGAACTCCAGTC * A * C-3'。


*表示磷酸硫代磷酸酯键。/5Phos/表示磷酸化)使用热循环仪通过以下程序进行退火:


。95°C 2分钟,缓慢冷却至25°C(0.1°C / S),接着在25°C 30 分钟,将退火后的适配器(50 MyuM )用DW 稀释成0.1 Myu M,并保存在- 20°C。


 


致谢


 


这项工作得到了 JST CREST JPMJCR15O2到AJN。此协议是根据长野等人(2015年)中描述的方法修改的。






利益争夺


 


我们没有利益冲突或利益冲突。


 


参考文献


 


Alpern ,D.,Gardeux ,V.,Russeil ,J.,Mangeat ,B.,Meireles-Filho ,ACA,Breysse ,R。,Hacker,D.和Deplancke ,B.(2019)。BRB -seq: ultra- 通过批量RNA条形码和测序实现的可负担得起的高通量转录组学。Genome Biol 20(1):71。
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Muir,P.,Li,S.,Lou,S.,Wang,D.,Spakowicz ,DJ,Salichos ,L。,Zhang,J.,Weinstock,GM,Isaacs,F.,Rozowsky ,J。和Gerstein, M.(2016)。测序的实际成本:扩展计算以与数据生成保持同步。Genome Biol 17:53 。
Nagano,AJ,Honjo ,MN,Mihara ,M。,Sato,M.和Kudoh,H.(2015)。使用RNA测序方法在自然环境中检测植物病毒 Mol Biol 1236:89-98。
Townsley ,BT,Covington,MF,Ichihashi ,Y.,Zumstein ,K。和Sinha,NR(2015).BrAD-seq:呼吸适配器定向测序:用于链特异性mRNA库的简化,超简单和快速的库制备方案。施工前植物科学6:366。
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Copyright Kashima 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. Kashima, M., Deguchi, A., Tezuka, A. and Nagano, A. J. (2020). Low-cost and Multiplexable Whole mRNA-Seq Library Preparation Method with Oligo-dT Magnetic Beads for Illumina Sequencing Platforms. Bio-protocol 10(12): e3496. DOI: 10.21769/BioProtoc.3496.
  2. Ishikawa, T., Kashima, M., Nagano, A. J., Ishikawa-Fujiwara, T., Kamei, Y., Todo, T. and Mori, K. (2017). Unfolded protein response transducer IRE1-mediated signaling independent of XBP1 mRNA splicing is not required for growth and development of medaka fish. eLife 6: e26845.
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