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Related Concept Videos

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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Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
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Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific...
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Related Experiment Video

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An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing
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Rapid and sensitive single-cell RNA sequencing with SHERRY2.

Lin Di1,2,3,4, Bo Liu5,6, Yuzhu Lyu1

  • 1School of Basic Medical Sciences, Beijing Key Laboratory of Neural Regeneration and Repair, Advanced Innovation Center for Human Brain Protection, Capital Medical University, Beijing, 100069, China.

BMC Biology
|September 29, 2022
PubMed
Summary

SHERRY2 offers a sensitive and accurate single-cell RNA sequencing (scRNA-seq) method without second-strand cDNA synthesis. This protocol enhances transcript quantification for low-abundance and difficult-to-amplify targets, improving biological discovery.

Keywords:
RNA-seqSingle cellTn5 transposase

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Current single-cell RNA sequencing (scRNA-seq) methods rely on full-length double-stranded complementary DNA synthesis and enrichment.
  • These methods face challenges in accurately quantifying low-abundance transcripts or those difficult to amplify fully.

Purpose of the Study:

  • To introduce SHERRY2, an optimized protocol for scRNA-seq that bypasses second-strand cDNA synthesis.
  • To improve sensitivity, accuracy, and reduce bias in scRNA-seq.

Main Methods:

  • Utilizes Tn5 transposase for direct cut-and-tag of DNA/RNA hetero-duplexes.
  • Eliminates pre-amplification and sequence-dependent bias.
  • Optimized for single nuclei and high-throughput applications.

Main Results:

  • SHERRY2 demonstrates significantly higher sensitivity and accuracy compared to other scRNA-seq methods, even for single nuclei.
  • Accurate quantification of transcription factors and long non-coding RNAs was achieved.
  • Bias-free results revealed enriched genes in specific cellular components and functions, outperforming other protocols.
  • Confirmed Myc expression dynamics in germinal center cells, previously only detectable by gene-specific methods.

Conclusions:

  • SHERRY2 provides high sensitivity, accuracy, and throughput for identifying numerous genes per cell.
  • The protocol can reveal subtle transcriptomic differences between cells.
  • Facilitates significant biological discoveries through improved single-cell transcriptomic profiling.