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RNA-seq03:21

RNA-seq

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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High-throughput Gene Tagging in Trypanosoma brucei
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High-Throughput Sequencing for Trypanosome Transcriptome Characterization.

Julius Mulindwa1, Kevin Leiss2, Christine Clayton3

  • 1Department of Biochemistry, College of Natural Sciences, Makerere University, Kampala, Uganda.

Methods in Molecular Biology (Clifton, N.J.)
|March 30, 2020
PubMed
Summary

This chapter provides RNASeq experimental design guidance for kinetoplastids. It includes parasite isolation, rRNA removal, and custom bioinformatics pipelines for transcriptomic analysis.

Keywords:
Gene expressionGene regulationMouse; TrypanosomaRNASeqRatRodentTranscriptomerRNA depletion

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Area of Science:

  • Molecular Biology
  • Bioinformatics
  • Parasitology

Background:

  • High-throughput sequencing of complementary DNA (RNA-Seq) is the preferred method for transcriptome analysis.
  • Kinetoplastids are a group of flagellated protozoan parasites with significant medical and veterinary importance.

Purpose of the Study:

  • To detail essential considerations for designing RNA-Seq experiments specifically for kinetoplastids.
  • To provide practical protocols for sample preparation and data analysis in kinetoplastid research.

Main Methods:

  • Protocols for isolating kinetoplastid parasites from rodent hosts.
  • Methods for ribosomal RNA (rRNA) removal from total RNA.
  • Development of custom bioinformatics pipelines for sequence alignment, data analysis, and visualization.

Main Results:

  • Established protocols for kinetoplastid parasite isolation and RNA extraction.
  • Demonstrated effective rRNA depletion for improved RNA-Seq data quality.
  • Presented functional custom pipelines for analyzing kinetoplastid transcriptomic data.

Conclusions:

  • Effective RNA-Seq experimental design and data analysis are crucial for kinetoplastid research.
  • The provided protocols and pipelines facilitate robust transcriptome analysis in these parasites.
  • This work supports advancements in understanding kinetoplastid biology and developing new control strategies.