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Page note from Luca: I've added some placeholders for CAGE and Other Methods in order to preserve the original formatting. I will take care of deleting them if they are not filled in a timely manner.

RNA-seq

As understood by [Luca Visentin]

At its most essential principle, RNA-seq is the practice of extracting and purifying RNA molecules from a set of cells (or even a single cell), and feed them to Next-Generation Sequencing (NGS). This technique allows us to harness incredibly in-depth information about the transcriptome, the complete set of transcripts of a cell. These include mRNA, rRNA, miRNA (micro RNA), non-coding RNA and other small RNA. Analyzing these data gives us insight on the complete transcriptome profile (in layman's terms, the set of genes which are transcribed at a certain time point, and their relative expression levels), exon content of transcripts, exon splicing, and gene fusion (the process where two genes fuse together to form a single transcript, and thus protein, which occurs often in cancer).

(Note: it is important to note that all steps involving RNA manipulation have to be carried out in RNase-free environments, including pipettes, pipette tips and various containers, as well as wearing gloves)

To perform RNA-seq, samples (which can span from tissues to single cells) are collected and RNA is extracted, typically using commercially-available kits such as RNAEasy (Quiagen Hilde) or TRIZOL (Life Technologies), among others. Pure RNA can be preserved for later use (by using kits such as RNAlater), enriched by size to isolate specific RNA types, such as miRNA by using miRVana chromatographic columns (Ambion), sequence-selected through probes, or enriched for specific features, such as a poly-A tail (for mRNA). Enriched RNA is then subjected to DNase digestion, to remove the often significant DNA contamination. (Note: I've added the kit's names just because they are very "imaginative")

Afterwards, samples are analysed for purity, protein contamination and RNA quantity, in order to obtain libraries of sufficient quality. Library preparation then occurs, where RNA is retro-transcribed into cDNA (which is more stable, important for the sequencing step) and subjected to several, NGS-platform-specific steps. The final result, however, is almost always PCR-enriched, adaptor-ligated, end-repaired, RNA-free ds DNA. At this point, the library can be stored, replicated or sent to sequence in NGS-equipped laboratories.

After NGS, huge amounts of data are obtained, and they need to be sieved through in silico. For more information on the techniques used at this step, I would refer you to the Bioinformatics course. It is sufficient to note that, though both alignment algorithms and statistics, all information discussed above can be harnessed. RNA-seq data is usually (especially for large studies) available online for free, and can be "mined" for additional information even after the original study has concluded. I link to some databases in the sources.

Sources

• First of all, the book "RNA-seq Data Analysis" by Eija Koperlaninen, Jarno Tuimala, Panu Somervuo, Mikael Huss, and Garry Wong; CRC Press (ISBN: 978-1-4665-9500-2)
• Wikipedia has a really good article on RNA-seq.
• Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009;10(1):57-63. (Link)
• For some information of the formulae behind RNA quantification through RNA-seq, some slides by the lectures of Colin Dewey, Mark Craven, and Anthony Gitter (link)
• More information on RNA extraction, as provided by Labome (link)
• The Gene Expression Atlas, curated by EBI, and the ENCODE project (for RNA-seq experiments), as examples of RNA-seq databases.

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Section change-log

• [20/03/2019 - 20:50] Luca Visentin: Created page.

GRO-seq

As understood by [Luca Visentin]

RNA-seq, although powerful, doesn't necessarily measure transcriptional activity of a certain locus, only the "snapshot" concentration of RNA present in the cell at a specific time point; of course, this quantity is due to transcription, but is also heavily influenced by RNA stability and turnover. GRO-seq, which stands for Global Run-On sequencing, has been a well-established method to assess de novo synthesis of RNA molecules, giving information about the transcriptional activity of a cell, rather than a transcriptome "snapshot".

Nuclei are isolated from cells (through ipotonic solutions that burst the cells), washed to remove nucleotides, and kept in ice-cold conditions. The cold blocks transcription, leaving Polymerase complexes blocked in their current position: this is key for the whole GRO-seq workflow. After transcription blockage, nuclei are then thawed and incubated at 30 °C together with brominated uracil (which have incorporated Bromine) and all other nucleotides. Elongation then resumes, incorporating these new labelled nucleotides in the newly born RNA. Formation of new PICs is prevented by the addition of the detergent sarkosyl (Sodium lauroyl sarcosinate), so only transcripts that were being transcribed at the moment of nuclear harvesting are studied. This step is called "Nuclear Run-On".

After some time, the nuclei are dissolved, RNA is extracted and immunoprecipitated (enriched) to select only Br-labelled RNA molecules using anti-BrdU antibodies. Once newly born RNA is isolated, RNA-seq steps for sample preparation and library creation are followed, and the created libraries are sequenced through NGS. The data thus created is analysed in silico, as per all other RNA-seq applications. 

To quote Alessandro Gardini (see Sources): "GRO-seq has shown unprecedented accuracy to ascertain defects in RNAPII elongation and pause-release as well as termination. [...] Additionally, GRO-seq has revealed that RNAPII fires bi-directionally at most mammalian promoters, initiating noncoding RNAs that are transcribed antisense with respect to the messenger RNA. Owing to their instability, these transcripts do not accumulate in the nucleus and elude most RNA detection protocols." It is redundant to say that this technique can provide exciting new insights on RNA transcription and regulation.

Sources

• Gardini, Alessandro. “Global Run-On Sequencing (GRO-Seq)” Methods in molecular biology (Clifton, N.J.) vol. 1468 (2017): 111-20. (PubMed Central link)
  
• 5-Bromouracil (Wikipedia)
• Sodium Lauroyl sarcosinate (Wikipedia)

Section Change-log

• [20/03/2019 - 21:30] Luca Visentin: Created Page.

CAGE

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Sources

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Section Change-log

• [20/03/2019 - 21:30] Luca Visentin: Created placeholder.

Other Techniques

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Section Change-log

• [20/03/2019 - 21:30] Luca Visentin: Created placeholder.