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RNA-Seq (named as an abbreviation of RNA sequencing) is a technique that uses next-generation sequencing to reveal the presence and quantity of RNA molecules in a biological sample, providing a snapshot of gene expression in the sample, also known as transcriptome. [2] [3]
The earliest RNA-Seq work was published in 2006 with one hundred thousand transcripts sequenced using 454 technology. [40] This was sufficient coverage to quantify relative transcript abundance. RNA-Seq began to increase in popularity after 2008 when new Solexa/Illumina technologies allowed one billion transcript sequences to be recorded.
The three main steps of sequencing transcriptomes of any biological samples include RNA purification, the synthesis of an RNA or cDNA library and sequencing the library. [16] The RNA purification process is different for short and long RNAs. [16] This step is usually followed by an assessment of RNA quality, with the purpose of avoiding ...
RNA Seq Experiment. The single-cell RNA-seq technique converts a population of RNAs to a library of cDNA fragments. These fragments are sequenced by high-throughput next generation sequencing techniques and the reads are mapped back to the reference genome, providing a count of the number of reads associated with each gene. [13]
For example, to investigate a biological process which is estimated to occur for an hour, a researcher might design an experiment where the process is triggered for five minutes, 15 minutes, 30 minutes, 45 minutes, one hour, and two hours in separate cell culture samples before harvesting the cells for RNA-seq analysis.
Single-cell RNA sequencing (scRNA-seq) provides the expression profiles of individual cells and is considered the gold standard for defining cell states and phenotypes as of 2020. [44] Although it is impossible to obtain complete information on every RNA expressed by each cell, due to the small amount of material available, gene expression ...
Cross-linking and immunoprecipitation (CLIP, or CLIP-seq) is a method used in molecular biology that combines UV crosslinking with immunoprecipitation in order to identify RNA binding sites of proteins on a transcriptome-wide scale, thereby increasing our understanding of post-transcriptional regulatory networks.
Rapid amplification of cDNA ends (RACE) is a technique used in molecular biology to obtain the full length sequence of an RNA transcript found within a cell. RACE results in the production of a cDNA copy of the RNA sequence of interest, produced through reverse transcription, followed by PCR amplification of the cDNA copies (see RT-PCR).