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  • 1.
    Danielsson, Frida
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Skogs, Marie
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Huss, Mikael
    Rexhepaj, Elton
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    O'Hurley, Gillian
    Klevebring, Daniel
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Pontén, Fredrik
    Gad, Annica K. B.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Majority of differentially expressed genes are down-regulated during malignant transformation in a four-stage model2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 17, p. 6853-6858Article in journal (Refereed)
    Abstract [en]

    The transformation of normal cells to malignant, metastatic tumor cells is a multistep process caused by the sequential acquirement of genetic changes. To identify these changes, we compared the transcriptomes and levels and distribution of proteins in a four-stage cell model of isogenically matched normal, immortalized, transformed, and metastatic human cells, using deep transcriptome sequencing and immunofluorescence microscopy. The data show that similar to 6% (n = 1,357) of the human protein-coding genes are differentially expressed across the stages in the model. Interestingly, the majority of these genes are down-regulated, linking malignant transformation to dedifferentiation. The up-regulated genes are mainly components that control cellular proliferation, whereas the down-regulated genes consist of proteins exposed on or secreted from the cell surface. As many of the identified gene products control basic cellular functions that are defective in cancers, the data provide candidates for follow-up studies to investigate their functional roles in tumor formation. When we further compared the expression levels of four of the identified proteins in clinical cancer cohorts, similar differences were observed between benign and cancer cells, as in the cell model. This shows that this comprehensive demonstration of the molecular changes underlying malignant transformation is a relevant model to study the process of tumor formation.

  • 2.
    Fagerberg, Linn
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Oksvold, Per
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Skogs, Marie
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Älgenäs, C.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Pontén, F.
    Sivertsson, Åsa
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Odeberg, Jacob
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Klevebring, Daniel
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kampf, C.
    Asplund, A.
    Sjöstedt, E.
    Al-Khalili Szigyarto, C.
    Edqvist, P. -H
    Olsson, I.
    Rydberg, U.
    Hudson, P.
    Ottosson Takanen, J.
    Berling, H.
    Björling, Lisa
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Tegel, Hanna
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Rockberg, J.
    Nilsson, Peter
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Navani, S.
    Jirström, K.
    Mulder, J.
    Schwenk, Jochen M.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zwahlen, Martin
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hober, Sophia
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Forsberg, Mattias
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Von Feilitzen, Kalle
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Contribution of antibody-based protein profiling to the human chromosome-centric proteome project (C-HPP)2013In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 12, no 6, p. 2439-2448Article in journal (Refereed)
    Abstract [en]

    A gene-centric Human Proteome Project has been proposed to characterize the human protein-coding genes in a chromosome-centered manner to understand human biology and disease. Here, we report on the protein evidence for all genes predicted from the genome sequence based on manual annotation from literature (UniProt), antibody-based profiling in cells, tissues and organs and analysis of the transcript profiles using next generation sequencing in human cell lines of different origins. We estimate that there is good evidence for protein existence for 69% (n = 13985) of the human protein-coding genes, while 23% have only evidence on the RNA level and 7% still lack experimental evidence. Analysis of the expression patterns shows few tissue-specific proteins and approximately half of the genes expressed in all the analyzed cells. The status for each gene with regards to protein evidence is visualized in a chromosome-centric manner as part of a new version of the Human Protein Atlas (www.proteinatlas.org).

  • 3.
    Klevebring, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology.
    On Transcriptome Sequencing2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis is about the use of massive DNA sequencing to investigate the transcriptome. During recent decades, several studies have made it clear that the transcriptome comprises a more complex set of biochemical machinery than was previously believed. The majority of the genome can be expressed as transcripts; and overlapping and antisense transcription is widespread. New technologies for the interroga- tion of nucleic acids have made it possible to investigate such cellular phenomena in much greater detail than ever before. For each application, special requirements need to be met. The work presented in this thesis focuses on the transcrip- tome and the development of technology for its analysis. In paper I, we report our development of an automated approach for sample preparation. The procedure was benchmarked against a publicly available reference data set, and we note that our approach outperformed similar manual procedures in terms of reproducibility. In the work reported in papers II-IV, we used different massive sequencing technologies to investigate the transcriptome. In paper II we describe a concatemerization approach that increased throughput by 65% using 454 sequencing,and we identify classes of transcripts not previously described in Populus. Papers III and IV both report studies based on SOLiD sequencing. In the former, we investigated transcripts and proteins for 13% of the human gene and detected a massive overlap for the upper 50% transcriptional levels. In the work described in paper IV, we investigated transcription in non-genic regions of the genome and detected expression from a high number of previ- ously unknown loci.

  • 4.
    Klevebring, Daniel
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Bjursell, Magnus
    KTH, School of Biotechnology (BIO), Gene Technology.
    Emanuelsson, Olof
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    In-Depth Transcriptome Analysis Reveals Novel TARs and Prevalent Antisense Transcription in Human Cell Lines2010In: PLOS ONE, ISSN 1932-6203, Vol. 5, no 3, p. e9762-Article in journal (Refereed)
    Abstract [en]

    Several recent studies have indicated that transcription is pervasive in regions outside of protein coding genes and that short antisense transcripts can originate from the promoter and terminator regions of genes. Here we investigate transcription of fragments longer than 200 nucleotides, focusing on antisense transcription for known protein coding genes and intergenic transcription. We find that roughly 12% to 16% of all reads that originate from promoter and terminator regions, respectively, map antisense to the gene in question. Furthermore, we detect a high number of novel transcriptionally active regions (TARs) that are generally expressed at a lower level than protein coding genes. We find that the correlation between RNA-seq data and microarray data is dependent on the gene length, with longer genes showing a better correlation. We detect high antisense transcriptional activity from promoter, terminator and intron regions of protein-coding genes and identify a vast number of previously unidentified TARs, including putative novel EGFR transcripts. This shows that in-depth analysis of the transcriptome using RNA-seq is a valuable tool for understanding complex transcriptional events. Furthermore, the development of new algorithms for estimation of gene expression from RNA-seq data is necessary to minimize length bias.

  • 5.
    Klevebring, Daniel
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Bjursell, Magnus K
    KTH, School of Biotechnology (BIO), Gene Technology.
    Emanuelsson, Olof
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    In-depth transcriptome analysis reveals novel TARs and prevalent antisense transcription in human cell lines.Manuscript (preprint) (Other academic)
    Abstract [en]

    Several recent studies have indicated that transcription is pervasive in regions outside of protein coding genes and that short antisense transcripts can originate from the promoter and terminator regions of genes. Here we investigate transcription of fragments longer than 200 nucleotides, focusing on antisense transcription for known protein coding genes and intergenic transcription. We find that roughly 12% to 16% of all reads that originate from promoter and terminator regions, respectively, map antisense to the gene in question. Furthermore, we detect a high number of novel transcriptionally active regions (TARs) that are generally expressed at a lower level than protein coding genes. We also investigate the correlation between RNA-seq data and microarray data and conclude that the correlation is dependant on gene length such that longer genes show a better correlation.

  • 6.
    Klevebring, Daniel
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Fagerberg, Linn
    KTH, School of Biotechnology (BIO), Proteomics.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics.
    Emanuelsson, Olof
    KTH, School of Biotechnology (BIO), Gene Technology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Analysis of transcript and protein overlap in a human osteosarcoma cell line2010In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 11, no 1, p. 684-Article in journal (Refereed)
    Abstract [en]

    Background: An interesting field of research in genomics and proteomics is to compare the overlap between the transcriptome and the proteome. Recently, the tools to analyse gene and protein expression on a whole-genome scale have been improved, including the availability of the new generation sequencing instruments and high-throughput antibody-based methods to analyze the presence and localization of proteins. In this study, we used massive transcriptome sequencing (RNA-seq) to investigate the transcriptome of a human osteosarcoma cell line and compared the expression levels with in situ protein data obtained in-situ from antibody-based immunohistochemistry (IHC) and immunofluorescence microscopy (IF).

    Results: A large-scale analysis based on 2749 genes was performed, corresponding to approximately 13% of the protein coding genes in the human genome. We found the presence of both RNA and proteins to a large fraction of the analyzed genes with 60% of the analyzed human genes detected by all three methods. Only 34 genes (1.2%) were not detected on the transcriptional or protein level with any method. Our data suggest that the majority of the human genes are expressed at detectable transcript or protein levels in this cell line. Since the reliability of antibodies depends on possible cross-reactivity, we compared the RNA and protein data using antibodies with different reliability scores based on various criteria, including Western blot analysis. Gene products detected in all three platforms generally have good antibody validation scores, while those detected only by antibodies, but not by RNA sequencing, generally consist of more low-scoring antibodies.

    Conclusion: This suggests that some antibodies are staining the cells in an unspecific manner, and that assessment of transcript presence by RNA-seq can provide guidance for validation of the corresponding antibodies.

  • 7.
    Klevebring, Daniel
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Gry, Marcus
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lindberg, Johan
    KTH, School of Biotechnology (BIO), Gene Technology.
    Eidefors, Anna
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Automation of cDNA Synthesis and Labelling Improves Reproducibility2009In: Journal of Biomedicine and Biotechnology, ISSN 1110-7243, E-ISSN 1110-7251, Vol. 2009, p. 396808-Article in journal (Refereed)
    Abstract [en]

    Background. Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads. Results. The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures. Conclusions. We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies.

  • 8.
    Klevebring, Daniel
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Street, Nathaniel
    Fahlgren, Noah
    Kasschau, Kristin D.
    Carrington, James C.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Jansson, Stefan
    Genome-wide profiling of Populus small RNAs2009In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 10, p. Article number 620-Article in journal (Refereed)
    Abstract [en]

    Background: Short RNAs, and in particular microRNAs, are important regulators of gene expression both within defined regulatory pathways and at the epigenetic scale. We investigated the short RNA (sRNA) population (18-24 nt) of the transcriptome of green leaves from the sequenced Populus trichocarpa using a concatenation strategy in combination with 454 sequencing. Results: The most abundant size class of sRNAs were 24 nt and these were generally associated with a number of classes of retrotransposons and repetitive elements. Some repetitive elements were also associated with 22 nt RNAs. We identified an sRNA hot-spot on chromosome 19, overlapping a region containing both the sex-determining loci and a major cluster of NBS-LRR genes. A number of phased siRNA loci were identified, a subset of which are predicted to target PPR and NBS-LRR disease resistance genes, classes of genes that have been significantly expanded in Populus. Additional loci enriched for sRNA production were identified. We identified 15 novel predicted microRNAs (miRNAs), including miRNA∗ sequences, and identified a novel locus that may encode a dual miRNA or a miRNA and short interfering RNAs (siRNAs). Conclusions: The short RNA population of P. trichocarpa is at least as complex as that of Arabidopsis. We provide a first genome-wide view of short RNA production for P. trichocarpa and identify new, non-conserved miRNAs.

  • 9.
    Lundberg, Emma
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Fagerberg, Linn
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Klevebring, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology.
    Matic, Ivan
    Geiger, Tamar
    Cox, Juergen
    Älgenäs, Cajsa
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Lundeberg, Joakim
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Mann, Matthias
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Defining the transcriptome and proteome in three functionally different human cell lines2010In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 6, p. 450-Article in journal (Refereed)
    Abstract [en]

    An essential question in human biology is how cells and tissues differ in gene and protein expression and how these differences delineate specific biological function. Here, we have performed a global analysis of both mRNA and protein levels based on sequence-based transcriptome analysis (RNA-seq), SILAC-based mass spectrometry analysis and antibody-based confocal microscopy. The study was performed in three functionally different human cell lines and based on the global analysis, we estimated the fractions of mRNA and protein that are cell specific or expressed at similar/different levels in the cell lines. A highly ubiquitous RNA expression was found with > 60% of the gene products detected in all cells. The changes of mRNA and protein levels in the cell lines using SILAC and RNA ratios show high correlations, even though the genome-wide dynamic range is substantially higher for the proteins as compared with the transcripts. Large general differences in abundance for proteins from various functional classes are observed and, in general, the cell-type specific proteins are low abundant and highly enriched for cell-surface proteins. Thus, this study shows a path to characterize the transcriptome and proteome in human cells from different origins.

  • 10.
    Lundin, Sverker
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Stranneheim, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Pettersson, Erik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Klevebring, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Increased Throughput by Parallelization of Library Preparation for Massive Sequencing2010In: PLOS ONE, ISSN 1932-6203, Vol. 5, no 3, p. e10029-Article in journal (Refereed)
    Abstract [en]

    Background: Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing. Methodology/Principal Findings: In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling. Conclusions/Significance: We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsize sequencing centers.

  • 11.
    Rimini, Rebecca
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics.
    Sundberg, Marten
    Sjöberg, Ronald
    Klevebring, Daniel
    Gry, Marcus
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics.
    Validation of serum protein profiles by a dual antibody array approach2009Article in journal (Refereed)
    Abstract [en]

    In recent years, affinity-based technologies have become important tools for serum profiling to uncover protein expression patterns linked to disease state or therapeutic effects. In this study, we describe a path towards the production of an antibody microarray to allow protein profiling of biotinylated human serum samples with reproducible sensitivity in the picomolar range. With the availability of growing numbers of affinity reagents, protein profiles are to be validated in efficient manners and we describe a cross-platform strategy based on data concordance with a suspension bead array to interrogate the identical set of antibodies with the same cohort of serum samples. Comparative analysis enabled to screen for high-performing antibodies, which were displaying consistent results across the two platforms and targeting known serum components. Moreover, data processing methods such as sample referencing and normalization were evaluated for their effects on inter-platform agreement. Our work suggests that mutual validation of protein expression profiles using alternative microarray platforms holds great potential in becoming an important and valuable component in affinity-based high-throughput proteomic screenings as it allows to narrow down the number of discovered targets prior to orthogonal, uniplexed validation approaches.

  • 12.
    Uhlén, Mathias
    et al.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Oksvold, Per
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Älgenäs, Cajsa
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Hamsten, Carl
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Fagerberg, Linn
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Klevebring, Daniel
    Department of Medical Epidemiology, Karolinska Institute.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Odeberg, Jacob
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Pontén, Fredrik
    Kondo, Tadashi
    Sivertsson, Åsa
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Antibody-based Protein Profiling of the Human Chromosome 212012In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, no 3Article in journal (Refereed)
    Abstract [en]

    A Human Proteome Project has been proposed to create a knowledgebased resource based on a systematical mapping of all human proteins, chromosome by chromosome, in a gene-centric manner. With this background, we here describe the systematic analysis of chromosome 21 using an antibody-based approach for protein profiling using both confocal microscopy and immunohistochemistry, complemented with transcript profiling using next generation sequencing data. We also describe a new approach for protein isoform analysis using a combination of antibody-based probing and isoelectric focusing. The analysis has identified several genes on chromosome 21 with no previous evidence on the protein level and the isoform analysis indicates that a large fraction of human proteins have multiple isoforms. A chromosome-wide matrix is presented with status for all chromosome 21 genes regarding subcellular localization, tissue distribution and molecular characterization of the corresponding proteins. The path to generate a chromosome-specific resource, including integrated data from complementary assay platforms, such as mass spectrometry and gene tagging analysis, is discussed.

1 - 12 of 12
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