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Related Concept Videos

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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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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.
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Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
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3' End Sequencing Library Preparation with A-seq2
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Full-Length Single-Cell RNA Sequencing with Smart-seq2.

Simone Picelli1

  • 1German Centre for Neurodegenerative Diseases (DZNE), Bonn, Germany. simone.picelli@iob.ch.

Methods in Molecular Biology (Clifton, N.J.)
|April 28, 2019
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Summary
This summary is machine-generated.

Single-cell RNA sequencing (scRNA-seq) reveals cellular differences. This guide details generating high-quality sequencing libraries using the sensitive Smart-seq2 method, addressing common challenges for accurate gene expression analysis.

Keywords:
AutomationFull-lengthHigh-throughputIn-house Tn5 transposaseNextera® XT kitRNA-seqSingle cellSmart-seq2Tagmentation

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Area of Science:

  • Molecular Biology
  • Genomics
  • Bioinformatics

Background:

  • Single-cell RNA sequencing (scRNA-seq) has revolutionized the study of cellular heterogeneity and gene expression.
  • Numerous scRNA-seq methods exist, differing in throughput, sensitivity, and scalability.
  • Full-length transcript profiling offers deeper insights than partial-length methods.

Purpose of the Study:

  • To provide a comprehensive guide for generating high-quality sequencing-ready libraries using the Smart-seq2 protocol.
  • To highlight common issues and pitfalls encountered during Smart-seq2 library preparation.
  • To offer practical solutions for optimizing data quality in scRNA-seq experiments.

Main Methods:

  • Detailed protocol for Smart-seq2 library preparation.
  • Troubleshooting common problems in library construction.
  • Quality control steps for sequencing-ready libraries.

Main Results:

  • Smart-seq2 is presented as a gold standard for full-length transcript profiling due to its sensitivity, precision, and scalability.
  • The chapter offers practical solutions to common challenges in scRNA-seq library preparation.
  • Emphasis on generating high-quality data for robust downstream analysis.

Conclusions:

  • Efficient generation of high-quality scRNA-seq libraries is crucial for accurate biological insights.
  • Smart-seq2 provides a robust and sensitive method for full-length transcript analysis.
  • This guide empowers researchers to overcome common obstacles and achieve reliable scRNA-seq data.