Transcriptional Characterization of Glioma Neural Stem Cells Diva ...

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
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