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Geo-seq is a method that utilizes both laser capture microdissection and single-cell RNA sequencing procedures to determine the spatial distribution of the transcriptome in tissue areas approximately ten cells in size. [17] The workflow involves removal and cryosectioning of tissue followed by laser capture microdissection.
FISSEQ combines the spatial context of RNA-FISH and the global transcriptome profiling of RNA-seq. [1] FISSEQ preserves the tissue allowing single molecule in situ RNA localization. The foundation of the method is a novel nucleic acid sequencing library construction method that stably cross-links cDNA amplicons within biological samples. [2]
The nucleotide sequences generated are typically around 100 bp in length, but can range from 30 bp to over 10,000 bp depending on the sequencing method used. RNA-Seq leverages deep sampling of the transcriptome with many short fragments from a transcriptome to allow computational reconstruction of the original RNA transcript by aligning reads ...
RNA selection/depletion: To analyze signals of interest, the isolated RNA can either be kept as is, enriched for RNA with 3' polyadenylated (poly(A)) tails to include only eukaryotic mRNA, depleted of ribosomal RNA (rRNA), and/or filtered for RNA that binds specific sequences (RNA selection and depletion methods table, below). RNA molecules ...
This method can be used for the co-mapping mRNA and protein levels at a near single-cell resolution in fresh or frozen formaldehyde-fixed tissue samples. DBiT-seq utilizes next generation sequencing (NGS) and microfluidics. This method allows for simultaneous spatial transcriptomic and proteomic analysis of a tissue sample.
RNA-seq is emerging (2013) as the method of choice for measuring transcriptomes of organisms, though the older technique of DNA microarrays is still used. [1] RNA-seq measures the transcription of a specific gene by converting long RNAs into a library of cDNA fragments. The cDNA fragments are then sequenced using high-throughput sequencing ...
Normalisation of RNA-seq data accounts for cell to cell variation in the efficiencies of the cDNA library formation and sequencing. One method relies on the use of extrinsic RNA spike-ins (RNA sequences of known sequence and quantity) that are added in equal quantities to each cell lysate and used to normalise read count by the number of reads ...
3' mRNA-seq is a quantitative, genome-wide transcriptomic technique based on the barcoding of the 3' untranslated region (UTR) of mRNA molecules. Unlike standard bulk RNA-seq, where short sequencing reads are generated along the entire length of mRNA transcripts, only the 3' end of polyadenylated RNAs are sequenced in 3' mRNA-seq.