Transcriptional Characterization of Glioma Neural Stem Cells Diva ...

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Chapter 5 Methods Contents 5.1 Tag-sequencing Data Processing . . . . . . . . . . . . . . . 94 5.2 Array Comparative Genomic Hybridization . . . . . . . . . 98 5.3 Differential Gene Expression . . . . . . . . . . . . . . . . . 100 5.4 Quantitative Real Time-PCR Validation . . . . . . . . . . . 101 5.5 Literature Mining . . . . . . . . . . . . . . . . . . . . . . . 107 5.6 Differential Isoform Expression . . . . . . . . . . . . . . . . 110 5.7 Differential Long ncRNA Expression . . . . . . . . . . . . . 112 5.8 Glioma Expression Signatures . . . . . . . . . . . . . . . . 112 5.9 External Dataset Expression Correlation . . . . . . . . . . 112 5.10 Glioblastoma Pathway Construction . . . . . . . . . . . . . 114 5.11 MicroRNA Target Prediction Analysis . . . . . . . . . . . . 117 5.1 Tag-sequencing Data Processing Instead of the traditional microarray expression profiling, Solexa sequencing was performed on total RNA from four GNS cell lines: G144ED, G144, G179 and G166, and two NS cell lines from fetal brain: CB541 and CB660 (Table 5.1). Tag-sequencing (Tag-seq) library preparation and sequencing was carried out using the Illumina DGE Tag Profiling kit according to the manufacturer’s instructions. We have submitted the data to ArrayExpress. The libraries were specifically constructed with the longSAGE protocol, in which first and second strand cDNA is synthesized with a biotinylated oligo deoxy-thymine (oligo-dT) onto streptavidin beads. The bound cDNA is cleaved at the 5’ end by an anchoring enzyme, such as NlaIII, that adds GTAC, the recognition site for MmeI, to the poly-thymine (poly-T) strand. Sequencing adapters with 94
5.1 Tag-sequencing Data Processing Methods Table 5.1: Summary of cell lines investigated with the Tag-seq platform. (M=Male, F=Female) Type of cell line Name of cell line Tissue Sex GNS G144 GBM M GNS G144ED GBM M GNS G166 GBM F GNS G179 Giant cell glioblastoma M NS CB541 Fetal forebrain - NS CB660 Fetal forebrain - an MmeI recognition site are then linked to the cDNA and MmeI digestion removes the 3’ portion of the cDNA from the beads, generating tags of con- stant length of 17nt that are then ligated to a 3’ adaptor and sequenced, as shown in figure 5.1 [490]. In the case of an Illumina sequencing platform, these Figure 5.1: Step by step diagram of the longSAGE protocol used to generate reads that contain tag sequences derived from the mRNA pool. 95
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Transcriptional Characterization of
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This dissertation is my own work an
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Acknowledgements I would like to th
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5.9 External Dataset Expression Cor
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Introduction 1
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1.1 Glial Cells in the Central Nerv
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1.2 Glioblastoma Multiforme Introdu
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1.3 Primary and Secondary Glioblast
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1.4 Pathways Involved in Glioblasto
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1.5 Pathway Crosstalk Introduction
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Chapter 2 Neurogenesis Contents 2.1
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2.1 Radial Glia Introduction VZ adj
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2.2 Neural Stem Cells Introduction
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6.7 Isoform Differential Expression
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6.8 Long ncRNA Differential Express
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6.8 Long ncRNA Differential Express
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Chapter 7 Dataset Correlation Analy
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7.1 Enrichment Analysis Results Tab
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7.1 Enrichment Analysis Results Fig
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7.1 Enrichment Analysis Results Tab
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7.2 Glioblastoma Expression Signatu
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7.3 Tumour Expression Correlation R
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7.4 Survival Analysis Results Table
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7.4 Survival Analysis Results 867 g
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7.5 Glioblastoma Pathway Analysis R
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8.1 Principles Results say, this is
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8.3 Databases Results Figure 8.1: O
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8.3 Databases Results Figure 8.3: W
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8.4 Filters Results Bayesian method
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8.5 Target Prediction Ensemble Anal
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9.1. Digital Profiling of GNS Cell
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9.2. MicroRNA Target Prediction Ana
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9.3. Concluding Remarks Appendix pr
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A.1 Differential Expression Appendi
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A.2 Classified Differential Express
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A.3 Quantitative RT-PCR Appendix Ta
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A.3 Quantitative RT-PCR Appendix We
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A.3 Quantitative RT-PCR Appendix Ta
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A.3 Quantitative RT-PCR Appendix We
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A.4 Tag-seq vs qRT-PCR Correlation
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Appendix "BEX5", "DIAPH2", "GBP3",
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End Select End If End Sub Appendix
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If NameStr.ToLower().Equals("query"
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Appendix C Long ncRNAs We detected
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C. Long ncRNAs Appendix Tag Accessi
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D.1 Pathway Interactions Appendix F
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D.2 Pathway Images Appendix Figure
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D.2 Pathway Images Appendix Figure
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E. Exon Array Data Appendix Average
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F. MicroRNA Array Data Appendix Tab
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F. MicroRNA Array Data Appendix GNS
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F. MicroRNA Array Data Appendix GNS
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Ln Natural logarithm LOH Loss of He
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3.3 Schematisation of cell cycle ph
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8.3 Workflows at the core of the pr
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7.1 Selected Gene Ontology terms an
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Bibliography [1] Cell signaling tec
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