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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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RNA Structure01:23

RNA Structure

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Overview
The basic structure of RNA consists of a five-carbon sugar and one of four nitrogenous bases. Although most RNA is single-stranded, it can form complex secondary and tertiary structures. Such structures play essential roles in the regulation of transcription and translation.
Different Types of RNA Have the Same Basic Structure
There are three main types of ribonucleic acid (RNA): messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). All three RNA types consist of a...
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RNA Stability01:53

RNA Stability

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Intact DNA strands can be found in fossils, while scientists sometimes struggle to keep RNA intact under laboratory conditions. The structural variations between RNA and DNA underlie the differences in their stability and longevity. Because DNA is double-stranded, it is inherently more stable. The single-stranded structure of RNA is less stable but also more flexible and can form weak internal bonds. Additionally, most RNAs in the cell are relatively short, while DNA can be up to 250 million...
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Ribosomal RNA Synthesis02:53

Ribosomal RNA Synthesis

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Ribosome synthesis is a highly complex and coordinated process involving more than 200 assembly factors. The synthesis and processing of ribosomal components occurs not only in the nucleolus but also in the nucleoplasm and the cytoplasm of eukaryotic cells.
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RNA Interference01:23

RNA Interference

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RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
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Alternative RNA Splicing02:18

Alternative RNA Splicing

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Alternative RNA splicing is the regulated splicing of exons and introns to produce different mature mRNAs from a single pre-mRNA. Unlike in constitutive splicing where a single gene produces a single type of mRNA, alternative splicing allows an organism to produce multiple proteins from a single gene and plays an important role in protein diversity.
There are five types of alternative RNA splicing that vary in the ways the pre-mRNA segments are removed or retained in the mature mRNA. The first...
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3' End Sequencing Library Preparation with A-seq2
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Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq.

Lorrayne Serra1, Dennis Z Chang2, Marissa Macchietto3

  • 1Department of Developmental and Cell Biology, University of California, Irvine, California, USA.

Bio-Protocol
|March 23, 2018
PubMed
Summary
This summary is machine-generated.

This study adapts Smart-seq2 for single-worm RNA sequencing, enabling gene expression analysis in individual nematodes. This method is valuable for studying gene expression variations in wild type and mutant nematode backgrounds.

Keywords:
C. elegansRNA-seqS. carpocapsaeTranscriptome

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Area of Science:

  • Molecular Biology
  • Genomics
  • Nematology

Background:

  • Most nematodes are too small for standard RNA sequencing (RNA-seq) without pooling many individuals.
  • This necessitates specialized techniques to study gene expression at the individual level.

Purpose of the Study:

  • To adapt the Smart-seq2 protocol for single-worm transcriptome sequencing.
  • To enable the study of gene expression variances in individual nematodes, including wild type and mutant strains.

Main Methods:

  • Adaptation of the Smart-seq2 protocol for individual nematode larvae and embryos.
  • Application to nematode species like *Steinernema carpocapsae* and *Caenorhabditis elegans*.
  • Utilizing the Galaxy online environment for RNA-seq data analysis.

Main Results:

  • Successful adaptation of Smart-seq2 for single-worm RNA sequencing.
  • Demonstrated applicability to *S. carpocapsae* and *C. elegans*.
  • Protocol is expected to be adaptable to other nematode species and small invertebrates.

Conclusions:

  • The adapted Smart-seq2 protocol provides a method for individual nematode transcriptome sequencing.
  • This technique facilitates the study of gene expression differences at the single-organism level.
  • The protocol and analysis pipeline are valuable tools for nematode research.