Optimization of Poplar mRNA purification for trancriptome library construction

Optimization of Poplar mRNA purification for trancriptome library construction RNA-Seq has quickly become the standard method for transcriptomics [1]. It is well understood that the quality of the sequencing data highly depends upon the quality of the library [2] and the size of target DNA fragments is a key parameter for RNA-Seq library construction [3]. As it is well-known, a typical workflow of an RNA-Seq assay involves extraction (and often further purification) of mRNA [4]. Thus isolation of high quality mRNA is very important for library construction and a number of protocols for mRNA purification have been further developed [5]. In our previous experiments, we found that different ratios and concentrations of purification reagents affect mRNA purification and thereby affecting the library quality. To find out the most suitable purification protocol for woody plants, we designed three purification strategies. The details of the procedures were described in Supplementary Materials. The first strategy was based on the purification protocol reported by Borodina and Ricarda Jost [6,7]. The second purification strategy was based on Dynabeads® mRNA Purification Kit for mRNA from total RNA preps (Catalog# 61005; Ambion®, Austin, USA). According to our laboratory experience and exploration, an additional purification step was added to the primary purification protocol. The third strategy was based on the second strategy but using higher concentrations of key purification reagents. The buffers and solutions used in the three purification strategies were shown in Table 1. Table 1. Buffers used in the three mRNA purification strategies   Buffer  Contents of the buffer  First strategy  Binding Buffer  20 mM Tris-HCl, pH 7.5, 1.0 M LiCl, 2 mM EDTA  Washing Buffer A  10 mM Tris-HCl, pH 7.5, 0.1 M LiCl, 1 mM EDTA, 0.1% LiDS  Lysis/Binding Buffer  —  Washing Buffer B  10 mM Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mM EDTA  Second strategy  Binding Buffer  20 mM Tris-HCl, pH 7.5, 2.5 M LiCl, 2 mM EDTA  Lysis/Binding Buffer  100 mM Tris-HCl, pH 7.5, 500 mM LiCl, 10 mM EDTA, 1% LiDS, 5 mM dithiothreitol (DTT)  Washing Buffer A  10 mM Tris-HCl, pH 7.5, 0.25 M LiCl, 1 mM EDTA, 0.5% LiDS  Washing Buffer B  10 mM Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mM EDTA  Third strategy  Binding Buffer  20 mM Tris-HCl, pH 7.5,5.0 M LiCl, 5 mM EDTA  Lysis/Binding Buffer  100 mM Tris-HCl, pH 7.5, 2.0 M LiCl, 10 mM EDTA, 5% LiDS, 5 mM dithiothreitol (DTT)  Washing Buffer A  10 mM Tris-HCl, pH 7.5, 1.0 M LiCl, 1 mM EDTA, 5% LiDS  Washing Buffer B  10 mM Tris-HCl, pH 7.5, 1.0 M LiCl, 1 mM EDTA    Buffer  Contents of the buffer  First strategy  Binding Buffer  20 mM Tris-HCl, pH 7.5, 1.0 M LiCl, 2 mM EDTA  Washing Buffer A  10 mM Tris-HCl, pH 7.5, 0.1 M LiCl, 1 mM EDTA, 0.1% LiDS  Lysis/Binding Buffer  —  Washing Buffer B  10 mM Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mM EDTA  Second strategy  Binding Buffer  20 mM Tris-HCl, pH 7.5, 2.5 M LiCl, 2 mM EDTA  Lysis/Binding Buffer  100 mM Tris-HCl, pH 7.5, 500 mM LiCl, 10 mM EDTA, 1% LiDS, 5 mM dithiothreitol (DTT)  Washing Buffer A  10 mM Tris-HCl, pH 7.5, 0.25 M LiCl, 1 mM EDTA, 0.5% LiDS  Washing Buffer B  10 mM Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mM EDTA  Third strategy  Binding Buffer  20 mM Tris-HCl, pH 7.5,5.0 M LiCl, 5 mM EDTA  Lysis/Binding Buffer  100 mM Tris-HCl, pH 7.5, 2.0 M LiCl, 10 mM EDTA, 5% LiDS, 5 mM dithiothreitol (DTT)  Washing Buffer A  10 mM Tris-HCl, pH 7.5, 1.0 M LiCl, 1 mM EDTA, 5% LiDS  Washing Buffer B  10 mM Tris-HCl, pH 7.5, 1.0 M LiCl, 1 mM EDTA  Total RNA was isolated from sterile plantlets of Poplar 84 K (Populus alba×Populus glandulosa) using the RNAprep Pure Plant Kit (Tiangen Company, Beijng, China) according to the manufacturer’s instructions and was quantified at OD260 and OD280 with a NanoDrop® spectrophotometer (NanoDrop Technologies, Wilmington, USA). The RNA integrity was assessed by the sharpness of ribosomal RNA bands visualized on a denaturing 1.2% agarose gel [8]. RNA integrity was further evaluated in an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, USA) [9]. All RNA molecules had distinct bands of 28S, 18S and 5.8S without degradation (Fig. 1). The A260/280 absorbance ratio was between 2.0 and 2.1, and the RIN value was above 8.0. Then mRNA was captured by the Dynabeads Oligo (dT)25 [10] and purified using the three strategies. The mRNA preparations obtained by three strategies were further analyzed by quality assessment of transcriptome library construction. Figure 1. View largeDownload slide Total RNA extracted from 84 K Poplar leaves, quality and quantity assessment using Agilent 2100 Bioanalyzer and agarose gel electrophoresis (A) The 18S-region and 28S-region cover the 18S peak and 28S peak, respectively. The 5S-region covers the small rRNA fragments (5S and 5.8S rRNA, and tRNA). The fast-region lies between the 5S-region and the 18S-region. The inter-region lies between the 18S-region and the 28S-region. And finally the post-region lies beyond the 28S-region. (B) Agarose gel electrophoresis of total RNA. Figure 1. View largeDownload slide Total RNA extracted from 84 K Poplar leaves, quality and quantity assessment using Agilent 2100 Bioanalyzer and agarose gel electrophoresis (A) The 18S-region and 28S-region cover the 18S peak and 28S peak, respectively. The 5S-region covers the small rRNA fragments (5S and 5.8S rRNA, and tRNA). The fast-region lies between the 5S-region and the 18S-region. The inter-region lies between the 18S-region and the 28S-region. And finally the post-region lies beyond the 28S-region. (B) Agarose gel electrophoresis of total RNA. The test results of the Agilent 2100 Bioanalyzer showed that mRNA obtained by the first mRNA purification strategy did not meet the requirement of on-machine sequencing because the fragment size and the center value were above 1000 bp (Supplementary Fig. S1A). The results of agarose gel electrophoresis of transcriptome libraries purified by the first option showed the library bands were too wide (Supplementary Fig. S1B–D). The results of the mRNA sample purified using the second mRNA purification strategy for constructing trancriptome libraries were shown in Fig. 2. It was found that the purification of mRNA affected the process of fragmentation, which further influences the final insert size of the library. Results showed that the second purification strategy could thoroughly remove the rRNA, tRNA and other impurities to get complete, high-purity mRNA molecules. Figure 2. View largeDownload slide Analysis of the transcriptome libraries prepared using mRNA obtained by the second purification strategy (A) Results from the Agilent 2100 Bioanalyzer. (B,C) Agarose gel electrophoresis. Figure 2. View largeDownload slide Analysis of the transcriptome libraries prepared using mRNA obtained by the second purification strategy (A) Results from the Agilent 2100 Bioanalyzer. (B,C) Agarose gel electrophoresis. The test results of the Agilent 2100 Bioanalyzer showed that mRNA obtained by the third mRNA purification strategy did not meet the requirement of on-machine sequencing. The fragment size was 300–400 b (the adapter not removed), and the concentration of the library was lower than the requirements of on-machine sequencing (Supplementary Fig. S2A). The results of agarose gel electrophoresis of transcriptome libraries purified by the third option showed the library bands were too narrow (Supplementary S2B,C). In summary, we have developed a high throughput technique of the combination of mRNA purification by binding to oligo (dT)25-linked magnetic beads that is an efficient method for the production of strand-specific mRNA-Seq libraries. When mRAN was contaminated with tRAN and rRNA, the effect of mRNA fragmentation was poor, because the best detection quality was 400–600 bp (with the adapter). Therefore, the first and third mRNA purification protocols did not meet the requirements of sequencing. The size of the target DNA fragments in the final library is a key parameter for RNA-Seq library construction, So the purification of mRNA has to be strictly controlled to ensure the quality of the library. The protocol we have developed will be useful for a broad range of construction of other transcriptome libraries. Supplementary Data Supplementary data are available at Acta Biochimica et Biophysica Sinica online. Funding This work was supported by the Fundamental Research Funds for the Central Universities (No. 2572016BA02), The 111 Project (No. B16010) and Harbin Applied Technology Research and Development Project (No. 2016RAQXJ065). References 1 Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet  2009, 10: 57– 63. Google Scholar CrossRef Search ADS PubMed  2 Metzker ML. Sequencing technologies—the next generation. Nat Rev Genet  2010, 11: 31– 46. Google Scholar CrossRef Search ADS PubMed  3 Griebel T, Zacher B, Ribeca P, Raineri E, Lacroix V, Guigó R, Sammeth M. Modelling and simulating generic RNA-Seq experiments with the flux simulator. Nucleic Acids Res  2012, 40: 10073– 10083. Google Scholar CrossRef Search ADS PubMed  4 Marine R, Polson SW, Ravel J, Hatfull G, Russell D, Sullivan M, Syed F, et al.  . Evaluation of a transposase protocol for rapid genera-tion of shotgun high-throughput sequencing libraries from nanogram quantitiesof DNA. Appl Environ Microbiol  2011, 77: 8071– 8079. Google Scholar CrossRef Search ADS PubMed  5 Archer N, Walsh MD, Shahrezaei V, Hebenstreit D. Modeling enzyme processivity reveals that RNA-Seq libraries are biased in characteristic and correctable ways. Cell Syst  2016, 3: 467– 479. Google Scholar CrossRef Search ADS PubMed  6 Borodina T, Adjaye J, Sultan M. A strand-specific library preparation protocol for RNA sequencing. Meth Enzymol  2011, 500: 79. Google Scholar CrossRef Search ADS PubMed  7 Jost R, Berkowitz O, Masle J. Magnetic quantitative reverse transcription PCR: a high-throughput method for mRNA extraction and quantitative reverse transcription PCR. Biotechniques  2007, 43: 206. Google Scholar CrossRef Search ADS PubMed  8 Karrer EE, Lincoln JE, Hogenhout S, Bennett AB, Bostock RM, Martineau B, Lucas WJ, et al.  . In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries. Proc Natl Acad Sci U S A  1995, 92: 3814– 3818. Google Scholar CrossRef Search ADS PubMed  9 Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, et al.  . The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol  2006, 7: 1– 14. Google Scholar CrossRef Search ADS PubMed  10 Jakobsen KS, Breivold E, Hornes E. Purification of mRNA directly from crude plant tissues in 15 minutes using magnetic oligo dT microspheres. Nucleic Acids Res  1990, 18: 3669. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Biochimica et Biophysica Sinica Oxford University Press

Optimization of Poplar mRNA purification for trancriptome library construction

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© The Author(s) 2018. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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Abstract

RNA-Seq has quickly become the standard method for transcriptomics [1]. It is well understood that the quality of the sequencing data highly depends upon the quality of the library [2] and the size of target DNA fragments is a key parameter for RNA-Seq library construction [3]. As it is well-known, a typical workflow of an RNA-Seq assay involves extraction (and often further purification) of mRNA [4]. Thus isolation of high quality mRNA is very important for library construction and a number of protocols for mRNA purification have been further developed [5]. In our previous experiments, we found that different ratios and concentrations of purification reagents affect mRNA purification and thereby affecting the library quality. To find out the most suitable purification protocol for woody plants, we designed three purification strategies. The details of the procedures were described in Supplementary Materials. The first strategy was based on the purification protocol reported by Borodina and Ricarda Jost [6,7]. The second purification strategy was based on Dynabeads® mRNA Purification Kit for mRNA from total RNA preps (Catalog# 61005; Ambion®, Austin, USA). According to our laboratory experience and exploration, an additional purification step was added to the primary purification protocol. The third strategy was based on the second strategy but using higher concentrations of key purification reagents. The buffers and solutions used in the three purification strategies were shown in Table 1. Table 1. Buffers used in the three mRNA purification strategies   Buffer  Contents of the buffer  First strategy  Binding Buffer  20 mM Tris-HCl, pH 7.5, 1.0 M LiCl, 2 mM EDTA  Washing Buffer A  10 mM Tris-HCl, pH 7.5, 0.1 M LiCl, 1 mM EDTA, 0.1% LiDS  Lysis/Binding Buffer  —  Washing Buffer B  10 mM Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mM EDTA  Second strategy  Binding Buffer  20 mM Tris-HCl, pH 7.5, 2.5 M LiCl, 2 mM EDTA  Lysis/Binding Buffer  100 mM Tris-HCl, pH 7.5, 500 mM LiCl, 10 mM EDTA, 1% LiDS, 5 mM dithiothreitol (DTT)  Washing Buffer A  10 mM Tris-HCl, pH 7.5, 0.25 M LiCl, 1 mM EDTA, 0.5% LiDS  Washing Buffer B  10 mM Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mM EDTA  Third strategy  Binding Buffer  20 mM Tris-HCl, pH 7.5,5.0 M LiCl, 5 mM EDTA  Lysis/Binding Buffer  100 mM Tris-HCl, pH 7.5, 2.0 M LiCl, 10 mM EDTA, 5% LiDS, 5 mM dithiothreitol (DTT)  Washing Buffer A  10 mM Tris-HCl, pH 7.5, 1.0 M LiCl, 1 mM EDTA, 5% LiDS  Washing Buffer B  10 mM Tris-HCl, pH 7.5, 1.0 M LiCl, 1 mM EDTA    Buffer  Contents of the buffer  First strategy  Binding Buffer  20 mM Tris-HCl, pH 7.5, 1.0 M LiCl, 2 mM EDTA  Washing Buffer A  10 mM Tris-HCl, pH 7.5, 0.1 M LiCl, 1 mM EDTA, 0.1% LiDS  Lysis/Binding Buffer  —  Washing Buffer B  10 mM Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mM EDTA  Second strategy  Binding Buffer  20 mM Tris-HCl, pH 7.5, 2.5 M LiCl, 2 mM EDTA  Lysis/Binding Buffer  100 mM Tris-HCl, pH 7.5, 500 mM LiCl, 10 mM EDTA, 1% LiDS, 5 mM dithiothreitol (DTT)  Washing Buffer A  10 mM Tris-HCl, pH 7.5, 0.25 M LiCl, 1 mM EDTA, 0.5% LiDS  Washing Buffer B  10 mM Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mM EDTA  Third strategy  Binding Buffer  20 mM Tris-HCl, pH 7.5,5.0 M LiCl, 5 mM EDTA  Lysis/Binding Buffer  100 mM Tris-HCl, pH 7.5, 2.0 M LiCl, 10 mM EDTA, 5% LiDS, 5 mM dithiothreitol (DTT)  Washing Buffer A  10 mM Tris-HCl, pH 7.5, 1.0 M LiCl, 1 mM EDTA, 5% LiDS  Washing Buffer B  10 mM Tris-HCl, pH 7.5, 1.0 M LiCl, 1 mM EDTA  Total RNA was isolated from sterile plantlets of Poplar 84 K (Populus alba×Populus glandulosa) using the RNAprep Pure Plant Kit (Tiangen Company, Beijng, China) according to the manufacturer’s instructions and was quantified at OD260 and OD280 with a NanoDrop® spectrophotometer (NanoDrop Technologies, Wilmington, USA). The RNA integrity was assessed by the sharpness of ribosomal RNA bands visualized on a denaturing 1.2% agarose gel [8]. RNA integrity was further evaluated in an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, USA) [9]. All RNA molecules had distinct bands of 28S, 18S and 5.8S without degradation (Fig. 1). The A260/280 absorbance ratio was between 2.0 and 2.1, and the RIN value was above 8.0. Then mRNA was captured by the Dynabeads Oligo (dT)25 [10] and purified using the three strategies. The mRNA preparations obtained by three strategies were further analyzed by quality assessment of transcriptome library construction. Figure 1. View largeDownload slide Total RNA extracted from 84 K Poplar leaves, quality and quantity assessment using Agilent 2100 Bioanalyzer and agarose gel electrophoresis (A) The 18S-region and 28S-region cover the 18S peak and 28S peak, respectively. The 5S-region covers the small rRNA fragments (5S and 5.8S rRNA, and tRNA). The fast-region lies between the 5S-region and the 18S-region. The inter-region lies between the 18S-region and the 28S-region. And finally the post-region lies beyond the 28S-region. (B) Agarose gel electrophoresis of total RNA. Figure 1. View largeDownload slide Total RNA extracted from 84 K Poplar leaves, quality and quantity assessment using Agilent 2100 Bioanalyzer and agarose gel electrophoresis (A) The 18S-region and 28S-region cover the 18S peak and 28S peak, respectively. The 5S-region covers the small rRNA fragments (5S and 5.8S rRNA, and tRNA). The fast-region lies between the 5S-region and the 18S-region. The inter-region lies between the 18S-region and the 28S-region. And finally the post-region lies beyond the 28S-region. (B) Agarose gel electrophoresis of total RNA. The test results of the Agilent 2100 Bioanalyzer showed that mRNA obtained by the first mRNA purification strategy did not meet the requirement of on-machine sequencing because the fragment size and the center value were above 1000 bp (Supplementary Fig. S1A). The results of agarose gel electrophoresis of transcriptome libraries purified by the first option showed the library bands were too wide (Supplementary Fig. S1B–D). The results of the mRNA sample purified using the second mRNA purification strategy for constructing trancriptome libraries were shown in Fig. 2. It was found that the purification of mRNA affected the process of fragmentation, which further influences the final insert size of the library. Results showed that the second purification strategy could thoroughly remove the rRNA, tRNA and other impurities to get complete, high-purity mRNA molecules. Figure 2. View largeDownload slide Analysis of the transcriptome libraries prepared using mRNA obtained by the second purification strategy (A) Results from the Agilent 2100 Bioanalyzer. (B,C) Agarose gel electrophoresis. Figure 2. View largeDownload slide Analysis of the transcriptome libraries prepared using mRNA obtained by the second purification strategy (A) Results from the Agilent 2100 Bioanalyzer. (B,C) Agarose gel electrophoresis. The test results of the Agilent 2100 Bioanalyzer showed that mRNA obtained by the third mRNA purification strategy did not meet the requirement of on-machine sequencing. The fragment size was 300–400 b (the adapter not removed), and the concentration of the library was lower than the requirements of on-machine sequencing (Supplementary Fig. S2A). The results of agarose gel electrophoresis of transcriptome libraries purified by the third option showed the library bands were too narrow (Supplementary S2B,C). In summary, we have developed a high throughput technique of the combination of mRNA purification by binding to oligo (dT)25-linked magnetic beads that is an efficient method for the production of strand-specific mRNA-Seq libraries. When mRAN was contaminated with tRAN and rRNA, the effect of mRNA fragmentation was poor, because the best detection quality was 400–600 bp (with the adapter). Therefore, the first and third mRNA purification protocols did not meet the requirements of sequencing. The size of the target DNA fragments in the final library is a key parameter for RNA-Seq library construction, So the purification of mRNA has to be strictly controlled to ensure the quality of the library. The protocol we have developed will be useful for a broad range of construction of other transcriptome libraries. Supplementary Data Supplementary data are available at Acta Biochimica et Biophysica Sinica online. Funding This work was supported by the Fundamental Research Funds for the Central Universities (No. 2572016BA02), The 111 Project (No. B16010) and Harbin Applied Technology Research and Development Project (No. 2016RAQXJ065). References 1 Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet  2009, 10: 57– 63. Google Scholar CrossRef Search ADS PubMed  2 Metzker ML. Sequencing technologies—the next generation. Nat Rev Genet  2010, 11: 31– 46. Google Scholar CrossRef Search ADS PubMed  3 Griebel T, Zacher B, Ribeca P, Raineri E, Lacroix V, Guigó R, Sammeth M. Modelling and simulating generic RNA-Seq experiments with the flux simulator. Nucleic Acids Res  2012, 40: 10073– 10083. Google Scholar CrossRef Search ADS PubMed  4 Marine R, Polson SW, Ravel J, Hatfull G, Russell D, Sullivan M, Syed F, et al.  . Evaluation of a transposase protocol for rapid genera-tion of shotgun high-throughput sequencing libraries from nanogram quantitiesof DNA. Appl Environ Microbiol  2011, 77: 8071– 8079. Google Scholar CrossRef Search ADS PubMed  5 Archer N, Walsh MD, Shahrezaei V, Hebenstreit D. Modeling enzyme processivity reveals that RNA-Seq libraries are biased in characteristic and correctable ways. Cell Syst  2016, 3: 467– 479. Google Scholar CrossRef Search ADS PubMed  6 Borodina T, Adjaye J, Sultan M. A strand-specific library preparation protocol for RNA sequencing. Meth Enzymol  2011, 500: 79. Google Scholar CrossRef Search ADS PubMed  7 Jost R, Berkowitz O, Masle J. Magnetic quantitative reverse transcription PCR: a high-throughput method for mRNA extraction and quantitative reverse transcription PCR. Biotechniques  2007, 43: 206. Google Scholar CrossRef Search ADS PubMed  8 Karrer EE, Lincoln JE, Hogenhout S, Bennett AB, Bostock RM, Martineau B, Lucas WJ, et al.  . In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries. Proc Natl Acad Sci U S A  1995, 92: 3814– 3818. Google Scholar CrossRef Search ADS PubMed  9 Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, et al.  . The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol  2006, 7: 1– 14. Google Scholar CrossRef Search ADS PubMed  10 Jakobsen KS, Breivold E, Hornes E. Purification of mRNA directly from crude plant tissues in 15 minutes using magnetic oligo dT microspheres. Nucleic Acids Res  1990, 18: 3669. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Journal

Acta Biochimica et Biophysica SinicaOxford University Press

Published: Feb 1, 2018

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