Transcription analysis and extragenic transcription Flashcards
Genome-wide transcription analysis and ncRNA regulation
What information might we want to find out about transcription?
- Which genes are transcribed in which cells/conditions?
- Where does transcription start and terminate for known genes?
- Are there additional transcription units not previously identified?
What methods can be used for genome-wide transcription analysis?
- RNA-seq
- RNA polII ChIP-seq
- GRO-seq
- NET-seq
RNA-seq method:
- Isolate RNA
- Convert it to a cDNA library
- Fragment cDNA
(or fragment RNA and convert to cDNA after) - Ligate adapter sequences onto cDNA fragments complementary to primers
- Amplify fragments using PCR
- Sequence fragments
- Map sequences onto the genome
What would we first do to cellular RNA when studying gene expression using RNA-seq?
Do a polyA selection so just mRNA is enriched to be isolated.
What results does RNA-seq give?
- Which genome sequences are transcribed (present in the RNA pool of a cell)
- How frequent this transcription is (abundance of RNA).
What do gaps in RNA-seq data read represent?
Introns - not present in mRNA.
RNA-seq advantages:
- Technically straightforward (can be done using a commerical kit or sequencing service)
- Gives information on stable / mature RNAs; allows us to see where splicing occurs and the splicing efficiency
RNA-seq disadvantages:
- Doesn’t retain strand information (don’t know which strand of cDNA was original mRNA)
- Unstable RNAs are not reported
What is stranded RNA-seq?
RNA-seq but retaining strand information (which strand of cDNA was mRNA).
How can we do stranded RNA-seq?
- Ligate the RNA adaptors prior to conversion to cDNA (5’ and 3’ adaptor always in the same orientation)
- Include dUTP in the second strand cDNA synthesis; allows us to then degrade second strand or avoid amplifying it.
What RNA-seq data implies a gene is transcribed?
Peaks on the genomic map. Level of transcription indicated by peak height.
What does an RNA-seq peak not corresponding to a gene mean?
It is a stable uncharacterised transcript (SUT).
RNA polII ChIP-seq method:
- Cross-link proteins to DNA
- Isolate chromatin
- Sonicate to fragment chromatin
- Incubate with RNA polII antibody
- Isolate the Ab-chromatin complexes
- Isolate the DNA from the complexes
- Ligate on adapters
- Amplify fragments using PCR
- Sequence fragments
- Map sequences onto the genome
RNA polII ChIP-seq advantages:
- Gives information on unstable RNAs
RNA polII ChIP-seq disadvantages:
- Prescence of of RNAPII does not necessarily prove functional transcription (polymerase may have stalled)
- No information on directionality (don’t know which way polymerase is transcribing)
What results does RNA polII ChIP-seq give?
- Which genome sequences are transcribed (regions of the genome that RNA polII is associated with)
- How frequent this transcription is (how much polymerase is associated with that region)
Why would we use different antibodies for RNA polII ChIP-seq?
The RNApolII tail can be dynamically modified.
What modification does RNA polII have during transcription initiation (promoter)?
Serine 5 phosphorylation
What modification does RNA polII have during transcription elongation (gene body)?
Serine 2 phosphorylation
GRO-seq method:
- Chill cells to arrest polymerases
- Lyse cells and isolate nuclei
- Incubate with bromoUTP labelled nucleotide and sarkosyl drug
- Increase temperature, allowing transcription to ‘run on’ using bromoUTP
- Newly transcribed RNA contains bromoUTP analog
- Use antibody to bromoUTP to pull down newly synthesised RNA
- Hydrolyse into small fragments
- Ligate on adapters
- Convert to cDNA
- Amplify fragments using PCR
- Sequence fragments
- Map sequences onto the genome
