DOCSLIB.ORG

In Vitro Recombination Cloning of Entire Cdna Libraries in Arabidopsis Thaliana and Its Application to the Yeast Two-Hybrid System

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
In Vitro Recombination Cloning of Entire Cdna Libraries in Arabidopsis Thaliana and Its Application to the Yeast Two-Hybrid System Funct Integr Genomics (2005) 5: 175–183 DOI 10.1007/s10142-005-0134-5 ORIGINAL PAPER Lukas Bürkle . Svenja Meyer . Hakan Dortay . Hans Lehrach . Alexander Heyl In vitro recombination cloning of entire cDNA libraries in Arabidopsis thaliana and its application to the yeast two-hybrid system Received: 16 June 2004 / Revised: 5 December 2004 / Accepted: 5 December 2004 / Published online: 16 February 2005 # Springer-Verlag 2005 Abstract In the postgenomic era many experiments rely acterized before and after the transfer into the expression on the availability of transcript sequence for cloning. As vector. However no significant difference could be de- these clones usually originate from cDNA libraries, the tected. The functionality of the in vitro recombination quality of these libraries is crucial. If a good library is system for the shuttling of entire libraries was then further generated it is desirable to use a versatile cloning system tested by protein-protein interaction screens. The results of suitable for many different kinds of applications. The the library characterization and of the yeast two-hybrid cloning systems based on in vitro recombination proves screens and their implications for large-scale proteomic fitting for this task. However, the use of this method for approaches are discussed. shuttling entire cDNA libraries between different vectors has not yet been studied in great detail. Here we describe Keywords Yeast two-hybrid . In vitro recombination the construction of four cDNA libraries from different tis- cloning . Arabidopsis . cDNA libraries sues of Arabidopsis thaliana, the shuttling of the libraries into expression vectors, and evaluation of this method as well as its suitability for downstream applications. Librar- Introduction ies were constructed from seedlings, hormone treated seedlings, flowers, developing seeds and primary leaves in The availability of complete genome sequences of a rap- the “entry vector” of the Gateway™ cloning system. After idly increasing number of species allows biological ques- initial characterization of the libraries, they were shuttled tions to be asked in greater complexity than ever before. into an expression vector (a yeast two-hybrid prey vector). Although it is exciting to do research in system biology, it To monitor for a size bias generally assumed to be inherent has to be grounded in the molecular-level understanding of to in vitro recombination methods, the libraries were char- the processes in question (for review see Kitano 2002). Unicellular organisms and animal model systems, like L. Bürkle . H. Lehrach . A. Heyl Escherichia coli, Saccharomyces cerevisiae, Caenorhab- Max Planck Institute for Molecular Genetics, ditis elegans,orDrosophila melanogaster have been the Ihnestrasse 73, focus of attention in the first of these large-scale ex- 14195 Berlin, Germany periments. However the sequencing of the model plants S. Meyer Arabidopsis and rice have enabled large-scale approaches RZPD, German Resource Centre for Genome Research, to also be conducted in plants (Arabidopsis Genome 2000; Heubnerweg 6, Bürkle et al. 2003; Fang et al. 2002; Goff et al. 2002; 14059 Berlin, Germany Kersten et al. 2002; Roberts 2002; Yu et al. 2002). The L. Bürkle need for large-scale investigation of biological processes Institute for Veterinary Biochemistry and Molecular Biology, becomes especially evident when considering the high University of Zurich, number of genes of unknown function and often inaccu- Winterthurerstrasse 190, rate bioinformatic predictions. For example, in A. thaliana 8057 Zurich, Switzerland the function of only 2,500 out of the roughly 28,000 genes H. Dortay . A. Heyl (*) have been investigated experimentally (Hilson et al. 2003) Institute for Biology, Applied Genetics, Free University Berlin, and whole genome transcription analysis reveals that up to Albrecht-Thaer-Weg 6, 30% of the genome is wrongly annotated (Yamada et al. 14195 Berlin, Germany e-mail: [email protected] 2003). As functional studies on the genomic level are Tel.: +49-30-83856550 dependent on the availability of full-length cDNA clones, Fax: +49-30-83854345 the quality of cDNA libraries as a source for those ORF 176 clones is critical. Thus the value of a cDNA library in- MS. After 5–7 days, when the cotyledons were fully ex- creases if its quality in terms of complexity and redun- panded, the MS was replaced by MS supplemented with dancy has been characterized. While cDNA libraries can appropriate plant hormones (1 μM abscisic acid, 1- be obtained from commercial and noncommercial sources, aminocyclopropane-1-carboxylic acid, benzyladenine, gib- most libraries available from stock centres are not charac- berellic acid, or α-naphthalene acetic acid; Sigma). After terized at all. 2 h 50% of the plants were harvested and frozen directly Another important factor in large-scale approaches is in liquid nitrogen, with the other 50% being collected after the cloning strategy to capture cDNA into vectors de- 24 h in the same manner. This should assure the presence signed for various purposes—a potentially time and re- of mRNA of primary response genes as well as of genes source consuming effort. Various methods have been upregulated after several hours in response to the hormone developed to circumvent this bottleneck, such as homol- treatment. For seed RNA isolation, siliques at all stages of ogous recombination in yeast or the application of in vitro development were harvested, frozen in liquid nitrogen and recombination systems (e.g. Creator™ or Gateway™). subsequently freeze-dried. The seeds were collected out of The great advantage of these cloning systems is the pos- the siliques by vacuum (Gibon et al. 2002). To isolate sibility to transfer the gene sequences of an entire library RNA from primary leaves, seeds were incubated for 2 quickly and easily from the original vector to different days at 4°C and then sown on soil. After 7–10 days of expression vector systems by in vitro recombination in a growth under standard conditions (see above) in the green- single tube reaction. This is particularly important as a house, the primary leaves of the seedlings were cut off good cDNA library can then be used in many different and collected in liquid nitrogen. For the flower mRNA, applications. inflorescences ranging from young flower buds to young To address both points, the aims of this study were siliques were harvested and immediately frozen in liquid twofold: (1) to create a valuable resource for plant mo- nitrogen. lecular biologists by generating high-quality and character- ized Arabidopsis cDNA libraries in the pENTR1A vector from several tissues using the Gateway™ system, and (2) RNA extraction to test the effectiveness of in vitro recombination systems for the shuttling of cDNA libraries used in large-scale The total RNA was extracted with TRIzol (Sigma) fol- approaches. Towards those aims, we constructed oligo- lowing the manufacturer’s manual. In short, the tissue was (dT) primed, Gateway-compatible Arabidopsis cDNA li- ground with mortar and pestle cooled by liquid nitrogen. braries representing RNA from seedlings, inflorescences, Of the ground tissue, 200 mg was incubated with 1 ml hormone-treated seedlings, primary leaves and developing TRIzol for 5 min at room temperature (RT). Cell debris seeds. To evaluate the suitability of the Gateway™ system was removed by centrifugation (12,000×g at 4°C for 10 for shuttling entire libraries, the cDNA libraries were min) and 0.4 ml chloroform was added. After vortexing characterized before and after the in vitro recombination the mix was incubated for 5 min at RT. The phases were step by restriction analysis and sequence examination of separated by centrifugation (12,000×g at 4°C for 15 min) randomly picked and sequenced clones. To assess the value and the aqueous phase was transferred to a new tube. A of the newly generated libraries for proteomic experiments, 0.6× volume of isopropyl alcohol and a 0.1× volume of 3 we chose four proteins of the Arabidopsis cytokinin signal M sodium acetate were added to this aqueous phase and transduction pathway and two other genes of interest as bait incubated for 10 min at 4°C. The precipitated RNA was proteins, which were subsequently screened for interaction pelleted by centrifugation (12,000×g at 4°C for 15 min) partners using the cDNA libraries. The quality of the and after the removal of the supernatant the RNA was generated cDNA libraries, the suitability of the in vitro washed twice with 70% ethanol. After drying, the RNA recombination system for entire cDNA libraries and the was resuspended in 50 μl di-ethylepyrocarbonate-treated results of the yeast two-hybrid screens are discussed. water. The quality and quantity of the RNA was checked by agarose gel and an Agilent biochip (data not shown). Materials and methods Construction of the cDNA libraries Plant material The cDNA libraries were constructed in collaboration with For the generation of the cDNA libraries, plants and the RZPD German Resource Center for Genome Re- tissues from A. thaliana, Col-0, were used. For seedlings, search, Heidelberg. In short, the total RNA was reverse- seeds were sterilized (in 50% ethanol, 25% H2O2, 25% transcribed using an oligo-(dT) primer containing a NotI H2O) and placed on Whatman paper, incubated for 2 days restriction site. A SalI adapter was ligated to the 5′ end of at 4°C in the dark, then grown until the cotyledons fully the first strand of the cDNA for second strand synthesis. expanded (5–7 days) at 26°C in 16 h light/8 h dark cycle The resulting cDNAs were cloned via the SalI/NotI sites on MS liquid medium. The seedlings were harvested and into the pENTR1A vector (Gateway, Invitrogen) by re- frozen directly in liquid nitrogen. For the hormone treat- striction and ligation reactions and subsequently trans- ment, seedlings were grown on Whatman paper soaked in formed into E.
Load more

Recommended publications
  • Proquesttm Pre-Made Cdna Libraries
    Instruction Manual TM ProQuest Pre-made cDNA Libraries For detecting protein-protein interactions Version B December 12, 2002 25-0617 Table of Contents General Information ..............................................................2 Overview...............................................................................5 Using ProQuest™ Libraries....................................................8 pPC86.................................................................................13 pEXP-AD502.......................................................................15 Recipe.................................................................................17 Accessory Products ............................................................18 Purchaser Notification.........................................................19 Technical Service................................................................22 References..........................................................................24 1 General Information Contents 2 x 0.5 ml aliquots and Storage Each cDNA library is supplied in 80% SOB medium, 20% (v/v) glycerol. Store the library at -80°C. The cDNA library is stable for six months when stored properly. Titer of the Each library has greater than 5 x 109 cfu (colony Libraries forming units) derived from > 107 primary clones to ensure complete representation of rare sequences. ProQuest™ The following ProQuest™ Pre-made cDNA Libraries are Pre-made available from Invitrogen. For more information on cDNA preparing the library, see page
    [Show full text]
  • In-Fusion Smarter Directional Cdna Library Construction Kit User Manual Table of Contents I
    Takara Bio USA In-Fusion® SMARTer® Directional cDNA Library Construction Kit User Manual Cat. No. 634933 (050421) Takara Bio USA, Inc. 1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: [email protected] United States/Canada Asia Pacific Europe Japan Page 1 of 40 800.662.2566 +1.650.919.7300 +33.(0)1.3904.6880 +81.(0)77.565.6999 In-Fusion SMARTer Directional cDNA Library Construction Kit User Manual Table of Contents I. List of Components .................................................................................................................................................. 4 II. Additional Materials Required .................................................................................................................................. 5 III. Introduction .......................................................................................................................................................... 6 IV. RNA Preparation & Handling ..............................................................................................................................11 A. Assessing the Quality of the RNA Template ........................................................................................................11 V. SMARTer cDNA Synthesis by LD-PCR .................................................................................................................13 A. Recommended Products ......................................................................................................................................13
    [Show full text]
  • Library Construction and Screening
    Library construction and screening • A gene library is a collection of different DNA sequences from an organism, • which has beenAlso called genomic libraries or gene banks. • cloned into a vector for ease of purification, storage and analysis. Uses of gene libraries • To obtain the sequences of genes for analysis, amplification, cloning, and expression. • Once the sequence is known probes, primers, etc. can be synthesized for further diagnostic work using, for example, hybridization reactions, blots and PCR. • Knowledge of a gene sequence also offers the possibility of gene therapy. • Also, gene expression can be used to synthesize a product in particular host cells, e.g. synthesis of human gene products in prokaryotic cells. two types of gene library depending upon the source of the DNA used. 1.genomic library. 2.cDNA library Types of GENE library: • genomic library contains DNA fragments representing the entire genome of an organism. • cDNA library contains only complementary DNA molecules synthesized from mRNA molecules in a cell. Genomic Library : • Made from nuclear DNA of an organism or species. • DNA is cut into clonable size pieces as randomly possible using restriction endonuclease • Genomic libraries contain whole genomic fragments including gene exons and introns, gene promoters, intragenic DNA,origins of replication, etc Construction of Genomic Libraries 1. Isolation of genomic DNA and vector. 2.Cleavage of Genomic DNA and vector by Restriction Endonucleases. 3.Ligation of fragmented DNA with the vector. 4.Transformation of
    [Show full text]
  • Global Tissue-Specific Transcriptome Analysis of Citrus Sinensis
    www.nature.com/scientificdata opeN Global tissue-specifc transcriptome Data DeScrIpTor analysis of Citrus sinensis fruit across six developmental stages Received: 9 May 2019 Guizhi Feng, Juxun Wu & Hualin Yi Accepted: 23 July 2019 Published: xx xx xxxx Citrus sinensis fruit is a type of nonclimacteric fruit that mainly consists of four tissues: the epicarp, albedo, segment membrane and juice sac. The fruit quality is determined by the characteristics of these four tissues. However, our knowledge of the molecular processes that occur in these four tissues during citrus fruit development and ripening is limited. Tissue-specifc transcriptomes provide a comprehensive and detailed molecular regulatory network of citrus fruit development and ripening. In our study, we collected four types of tissue from C. sinensis fruits at six developmental stages. A total of 72 libraries were constructed from 24 samples (each sample had three replicates), and the transcriptomes were sequenced by an Illumina HiSeq 4000. The comprehensive analyses of the transcriptomes from the four tissues and six developmental stages presented here provide a valuable resource for the discovery of the molecular networks underlying citrus fruit development and ripening. Background & Summary Citrus, a nonclimacteric fruit, is widely cultivated worldwide. Citrus is mainly composed of the inedible epicarp (EP) and albedo (AL) and the edible segment membrane (SM) and juice sac (JS). Te development and ripening of citrus fruit is a complex and sophisticated regulatory process that can be divided into three stages: cell division stage; expansion stage involving cell enlargement and water, sugar accumulation; and ripening stage1. At present, most studies investigating citrus fruit development and ripening are based on the organ-wide or mixed-tissue level, which inevitably obscures many tissue-specifc phenomena.
    [Show full text]
  • Combinatorial Cdna Library by Complementation of an Auxotrophic Escherichia Coli: Antibodies for Orotate Decarboxylation JEFFREY A
    Proc. Natd. Acad. Sci. USA Vol. 91, pp. 8319-8323, August 1994 Biochemistry Selection of catalytic antibodies for a biosynthetic reaction from a combinatorial cDNA library by complementation of an auxotrophic Escherichia coli: Antibodies for orotate decarboxylation JEFFREY A. SMILEY AND STEPHEN J. BENKOVIC* Department of Chemistry, The Pennsylvania State University, University Park, PA 16802 Contributed by Stephen J. Benkovic, May 23, 1994 ABSTRACT Antibodies capable of decarboxylating oro- tate were sought by immunization with a hapten designed to elicit antibodies with combining sites that resemble the orotate- binding and catalytic portion of the active site of the enzyme OPRTase ODCase orotidine 5'-monophosphate (OMP) decarboxylase (orotidine- 0 '00 \\ 5'-monophosphate carboxy-lyase, EC 4.1.1.23). Active recom- binant antibody fragments (Fabs) were selected from a com- HN 'PO4 binatorial cDNA library by complementation of a pyrF strain UMP ofEscherichia cofl and growth ofthe library-expressing cells on pyrimidine-free medium. In this biological screen, a suffi- H " 0 ciently active antibody from the library would decarboxylate Antibody \ Urasi orotate to produce uracil, a pyrimidine source for the aux- Catalysis PRTase otroph, and would provide the cells with a growth advantage compared to cells without an active antibody. Six recombinant AN Fabs yielded identifiable colonies in a screen of 16,000 trans- H formants. To enhance its stability and expression level, one of the six positive fragments was converted into single-chain form. FIG. 1. Pathways for UMP biosynthesis. Upper route involves In this form, the antibody a naturally occurring enzymes orotate phosphoribosyltransferase fragment conferred definite growth (OPRTase) and OMP decarboxylase (ODCase).
    [Show full text]
  • Efficient PCR Approach for the Selection of Cdnas Encoded In
    Proc. Nati. Acad. Sci. USA Vol. 88, pp. 9623-9627, November 1991 Genetics cDNA selection: Efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments (yeast artificial chromosomes/cosmids/major histocompatibility complex) SATISH PARIMOO*t, SANKHAVARAM R. PATANJALI*, HRIDAYABHIRANJAN SHUKLA*, DAVID D. CHAPLIN*, AND SHERMAN M. WEISSMAN* *Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510; and tDepartment of Internal Medicine and Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110 Contributed by Sherman M. Weissman, July 24, 1991 ABSTRACT Identification of coding segments in large B30H3, isolated by one of us (D.D.C.; ref. 20), encompasses fragments of genomic DNA is a recurrent problem in genome a 320-kilobase (kb) region from the human major histocom- mapping and positional cloning studies. We have developed a patibility complex (MHC) class I HLA-A locus. Yeast chro- rapid and efficient protocol to achieve this goal, based on mosomes from agarose plugs were fractionated by 1% aga- hybridization of cDNA fragments to immobilized DNA and rose contour-clamped homogeneous electric field gel elec- recovery of the selected cDNAs by the PCR. The procedure trophoresis (21) in 0.5x TBE (45 mM Tris HCl, pH 8.3/45 permits rapid cloning of cDNA fragments encoded by large mM boric acid/0.2 mM EDTA) at 14'C for 45 hr at 150 V/cm genomic DNA fragments, groups of yeast artificial chromo- with a switching interval of 30 sec in an LKB Pulsaphor somes, or cosmids and has the potential to directly enrich apparatus.
    [Show full text]
  • Manual: Lambda ZAP-CMV XR Library Construction
    Lambda ZAP-CMV XR Library Construction Kit INSTRUCTION MANUAL Catalog #200448 BN #200448-12 Revision A.01 For In Vitro Use Only 200448-12 LIMITED PRODUCT WARRANTY This warranty limits our liability to replacement of this product. No other warranties of any kind, express or implied, including without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided by Agilent. Agilent shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use this product. ORDERING INFORMATION AND TECHNICAL SERVICES United States and Canada Agilent Technologies Stratagene Products Division 11011 North Torrey Pines Road La Jolla, CA 92037 Telephone (858) 373-6300 Order Toll Free (800) 424-5444 Technical Services (800) 894-1304 Internet [email protected] World Wide Web www.stratagene.com Europe Location Telephone Fax Technical Services Austria 0800 292 499 0800 292 496 0800 292 498 Belgium 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 15775 0800 15740 0800 15720 France 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 919 288 0800 919 287 0800 919 289 Germany 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 182 8232 0800 182 8231 0800 182 8234 Netherlands 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 023 0446 +31 (0)20 312 5700 0800 023 0448 Switzerland 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 563 080 0800 563 082 0800 563 081 United Kingdom 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 917 3282 0800 917 3283 0800 917 3281 All Other Countries Please contact your local distributor.
    [Show full text]
  • Full-Length Mrna Sequencing Reveals Principles of Poly(A) Tail Length Control
    bioRxiv preprint doi: https://doi.org/10.1101/547034; this version posted February 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Full-length mRNA sequencing reveals principles of poly(A) tail length control Ivano Legnini*,1, Jonathan Alles*,1, Nikos Karaiskos1, Salah Ayoub1 and Nikolaus Rajewsky1,$ Laboratory for Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine, Robert-Rössle Str 10, Berlin-Buch, Germany * eQual contribution $ correspondence to [email protected] Summary Although mRNAs are key molecules for understanding life, there exists no method to determine the full-length seQuence of endogenous mRNAs including their poly(A) tails. Moreover, although poly(A) tails can be modified in functionally important ways, there also exists no method to accurately seQuence them. Here, we present FLAM-seQ, a rapid and simple method for high-Quality seQuencing of entire mRNAs. We report a cDNA library preparation method coupled to single-molecule seQuencing to perform FLAM-seQ. Using human cell lines, brain organoids, and C. elegans we show that FLAM-seQ delivers high-Quality full-length mRNA seQuences for thousands of different genes per sample. We find that (a) 3’ UTR length is correlated with poly(A) tail length, (b) alternative polyadenylation sites and alternative promoters for the same gene are linked to different tail lengths, (c) tails contain a significant number of cytosines. Thus, we provide a widely useful method and fundamental insights into poly(A) tail regulation.
    [Show full text]
  • DNA Libraries (PART-I)
    NPTEL – Biotechnology – Fundamentals of Biotechnology Lecture 10: DNA Libraries (PART-I) Introduction-Gene sequence are arranged in genome in a randon fashion and selecting or isolating a gene is a big task especially when the genomic sequences are not known. A small portion of genome is transcribed to give mRNA where as a major portion remained untranscribed. Hence, there are two ways to represent a genomic sequence information into the multiple small fragments in the form of a library: (1) Genomic library (2) cDNA library. Preparation of Genomic Library-A genomic library represents complete genome in multiple clones containing small DNA fragments. Depending upon organism and size of genome, this library is either prepared in a bacterial vector (discussed later in future lectures) or in yeast artificial chromosome (YAC). An outline of the construction of genomic library is given in Figure 10.1. it has following steps: 1. Isolation of genomic DNA 2. Generation of suitable size DNA fragments 3. Cloning in suitable vector system (depending on size) 4. Transformation in suitable host . Joint initiative of IITs and IISc – Funded by MHRD Page 1 of 65 NPTEL – Biotechnology – Fundamentals of Biotechnology Figure 10.1: Contruction of Genomic library. Joint initiative of IITs and IISc – Funded by MHRD Page 2 of 65 NPTEL – Biotechnology – Fundamentals of Biotechnology 1. Isolation of genomic DNA- Isolation of genomic DNA has following steps: 1. Lysis of cells with detergent containing lysis buffer. 2. Incubation of cells with digestion buffer containing protease-K, SDS to release genomic DNA from DNA-protein complex. 3. Isolation of genomic DNA by absolute alchol precipitation.
    [Show full text]
  • Human Thrombomodulin Gene Is Intron Depleted
    Proc. Natl. Acad. Sci. USA Vol. 84, pp. 6425-6429, September 1987 Biochemistry Human thrombomodulin gene is intron depleted: Nucleic acid sequences of the cDNA and gene predict protein structure and suggest sites of regulatory control (protein C/coated-pit receptors/endothelial cell) ROBERT W. JACKMAN*, DAVID L. BEELER*, LINDA FRITZE*t, GERALD SOFF*t, AND ROBERT D. ROSENBERG*t *Department of Biology and Whitaker College, Massachusetts Institute of Technology, Cambridge, MA 02139; and tthe Department of Medicine, Harvard Medical School and the Beth Israel Hospital, Boston, MA 02215 Communicated by Phillips W. Robbins, June 19, 1987 ABSTRACT We have isolated a human thrombomodulin METHODS cDNA, and a human genomic clone containing the putative promoter domain, as well as the translated and untranslated Screening of Human cDNA and Genomic Libraries. The regions of the endothelial cell receptor. The nucleotide se- human cDNA and genomic libraries were screened (3) on quence of the thrombomodulin cDNA allows us to provide a Escherichia coli Y 1088 or LE 392 (cDNA library), and LE complete picture of the structure of this endothelial cell 392 (genomic library) with Colony Plaque Screen (New receptor, and to confirm its homology to the human low density England Nuclear) according to the following modifications of lipoprotein receptor. The nucleotide sequence of the thrombo- the manufacturer's instructions. The filters were lifted in modulin gene suggests areas within the putative promoter duplicate, air-dried, autoclaved for 1 min, air-dried again, and domain that may be critical for regulating expression of the then baked at 80°C for 1 hr (4).
    [Show full text]
  • Sequencing of First-Strand Cdna Library Reveals Full-Length
    ARTICLE Received 20 Mar 2014 | Accepted 2 Dec 2014 | Published 21 Jan 2015 DOI: 10.1038/ncomms7002 Sequencing of first-strand cDNA library reveals full-length transcriptomes Saurabh Agarwal1, Todd S. Macfarlan2, Maureen A. Sartor3 & Shigeki Iwase1 Massively parallel strand-specific sequencing of RNA (ssRNA-seq) has emerged as a powerful tool for profiling complex transcriptomes. However, many current methods for ssRNA-seq suffer from the underrepresentation of both the 50 and 30 ends of RNAs, which can be attributed to second-strand cDNA synthesis. The 50 and 30 ends of RNA harbour crucial information for gene regulation; namely, transcription start sites (TSSs) and polyadenylation sites. Here we report a novel ssRNA-seq method that does not involve second-strand cDNA synthesis, as we Directly Ligate sequencing Adaptors to the First-strand cDNA (DLAF). This novel method with fewer enzymatic reactions results in a higher quality of the libraries than the conventional method. Sequencing of DLAF libraries followed by a novel analysis pipeline enables the profiling of both 50 ends and polyadenylation sites at near-base resolution. Therefore, DLAF offers the first genomics tool to obtain the ‘full-length’ transcriptome with a single library. 1 Department of Human Genetics, University of Michigan, Ann Arbor, Michigan 48109, USA. 2 Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA. 3 Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan 48109, USA. Correspondence and requests for materials should be addressed to S.I. (email: [email protected]). NATURE COMMUNICATIONS | 6:6002 | DOI: 10.1038/ncomms7002 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited.
    [Show full text]
  • SMART™ Cdna Library Construction Kit User Manual
    User Manual SMART™ cDNA Library Construction Kit User Manual United States/Canada 800.662.2566 Asia Pacific +1.650.919.7300 Europe +33.(0)1.3904.6880 Japan +81.(0)77.543.6116 Cat. No. 634901 Clontech Laboratories, Inc. PT3000-1 (PR043527) A Takara Bio Company 1290 Terra Bella Ave. Published May 2010 Mountain View, CA 94043 Technical Support (US) E-mail: [email protected] www.clontech.com SMART™ cDNA Library Construction Kit User Manual Table of Contents I. Introduction 4 II. List of Components 9 III. Additional Materials Required 12 IV. General Considerations 15 V. SMART cDNA Synthesis by LD PCR 18 A. First-Strand cDNA Synthesis 18 B. cDNA Amplification by LD PCR 19 C. Proteinase K Digestion 21 D. SfiI Digestion 21 E. cDNA Size Fractionation by CHROMA SPIN-400 22 VI. SMART cDNA Synthesis by Primer Extension 24 A. First-Strand cDNA Synthesis 24 B. ds cDNA Synthesis by Primer Extension 25 C. Proteinase K Digestion 26 D. SfiI Digestion 27 E. cDNA Size Fractionation by CHROMA SPIN-400 27 VII. SMART cDNA Library Protocols 30 A. Ligation of cDNA to λTriplEx2 Vector 30 B. Bacterial Culture Plating 31 C. Titering the Unamplified Library 32 D. Determining the Percentage of Recombinant Clones 33 E. Library Amplification 34 F. Titering the Amplified Library 35 VIII. Troubleshooting Guide 37 IX. References 42 Appendix A: Typical Results of ds cDNA Synthesis 43 Appendix B: Converting λTriplEx2 to pTriplEx2 45 Appendix C: Restriction Map of λTriplEx2 48 Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3000-1 2 Version No.
    [Show full text]