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High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)
Published on: October 5, 2018
High-resolution analysis of the 5'-end transcriptome using a next generation DNA sequencer.
Shin-ichi Hashimoto1, Wei Qu, Budrul Ahsan
1Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Plos One
|January 3, 2009
Summary
This study introduces a new 5'-end transcriptome workflow using the SOLiD system for cost-effective, high-sensitivity whole genome gene expression analysis. The method significantly improves sensitivity and dynamic range compared to traditional approaches.
Area of Science:
- Genomics
- Molecular Biology
- Bioinformatics
Background:
- Whole genome gene expression studies are crucial for understanding cellular processes.
- Traditional methods face limitations in sensitivity and dynamic range.
- Tag-based sequencing offers a promising alternative for high-throughput gene expression analysis.
Purpose of the Study:
- To develop and validate a 5'-end transcriptome workflow for the SOLiD system.
- To assess the sensitivity and dynamic range advantages of this tag-based approach.
- To investigate whole genome gene expression in a colon cancer cell line treated with a DNA methyltransferase inhibitor.
Main Methods:
- Development of a 5'-end transcriptome library preparation protocol for the SOLiD platform.
- Massively parallel sequencing of 25-base 5'-end tags from HT-29 colon cancer cells (untreated and 5-aza-2'-deoxycytidine treated).
- Bioinformatic analysis to map tags to the human genome and quantify gene expression levels.
Main Results:
- Generated over 20 million 5'-end tags, mapping to approximately 14,000 protein-coding genes.
- Achieved a dynamic range of gene expression from 0.02 to 4,704 transcripts per cell.
- Demonstrated 100-1,000 fold greater sensitivity compared to 5'end SAGE using Sanger sequencing.
Conclusions:
- The SOLiD-based 5'-end transcriptome workflow provides high-resolution gene expression profiling.
- This method offers significant improvements in sensitivity and dynamic range for whole genome expression studies.
- The findings offer insights into transcriptional regulation and cell biology, particularly in cancer contexts.
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