Biology Stack Exchange
Biology Stack Exchange

Questions tagged [rna-sequencing]

Determining the sequence of RNA. This technique is used for discovery and quantification of mRNAs and ncRNAs.

Involves library preparation and sequencing. Library preparation might slightly vary for different classes of RNAs. The general procedure for mRNA and most ncRNA sequencing involves:

  1. Enrichment of polyA RNAs using oligo-dT beads to remove ribosomal RNA. Alternatively, ribosomal RNAs can be selectively removed. Refer to this.
  2. cDNA preparation
  3. Adapter ligation
  4. PCR amplification
  5. Sequencing

See also .

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RNA seq and using of Poly(A) or non-Poly(A) based amplification of RNA

I'm studying "Deep sequencing the circadian and diurnal transcriptome of Drosophila brain" Hughes et al., 2012. I've got some problems with the materials and methods. Before RNAseq, the authors amplify RNA. They use two kits: Poly(A) based and…
user4564
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New GO terms after TopGO enrichment?

I ran an enrichment analysis with custom annotations using TopGO and surprisingly I obtained new GO terms inside the significant GO terms. Is that possible? The only reason I can imagine this is possible is because TopGO adds new GO terms based on…
biotech
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Metatranscriptomics Extraction

I am attempting to perform metatranscriptomics analysis of a cleanroom. The DNA input is known to be rather low (<1pg). Despite this, I want to still attempt to try it. I am thinking it might be best to perform extraction trials before hand where…
user84756
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RNA contains Uracil in place of Thymine

Thymine does not present in RNA but DNA containing thymine why
Rahul Daksh
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Understanding Illumina Adapters

I am currently working on a project where I need to trim the adapters off of some single end read RNA-Seq data, and I want to know which sequences to cleave. Illumina TruSeq adapters were used. I have tried following Tuft's explanation and make…
irritable_phd_syndrome
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Regarding the difference between cDNA library and RNA sequencing (Biochem. technique)

I was wondering why establishment of cDNA library required the step of reverse transcription (i.e. turning the sequence into DNA) why not directly using the extracted RNA for sequencing and converting the U to T base? Why bother to have one more…
patpat
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the meaning of "Non-primary hits" and "tags"

Can I ask you to help understand the meaning of "Non-primary hits" and "tags" in the following paragraph related to RSeQC: The result table reports total number of reads (excluding nonprimary hits) and tags (separate splice fragments of a read).…
omid
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3 end RNA seq library construction

I have tried to make cDNA libraries from FFPE tumor blocks (highly degraded RNA) using the 3' end RNA seq protocol provided by West lab. Here is the link for the same. http://med.stanford.edu/labs/vanderijn-west/Protocols.html After PCR enrichment,…
Manisha
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dbSNP clarification

I am currently going through this paper - link. In their methods section the authors have described they have used dbSNP for checking biallelic expression of genes. I would like to know if dbSNP contains SNPs for intergenic regions i.e. for non…
Roni Saiba
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