GIGUERE - LECTURE 4 Flashcards
Techniques to study mechanisms of transcription
how did maxam and gilbert sequence DNA
how did sanger sequence DNA
how did high throughput DNA sequencing 1st generation work?
how did high throughput DNA sequencing 2nd generation work?
how did high throughput DNA sequencing 3rd generation work?
which animal has the largest genome
what is the transcriptome
the complete set of RNA transcripts present in a given cell type, and their quantity for a specific developmental stage, physiological condition or disease state
what are the aims of transcriptomics
how does RNA-seq work
what are some benefits/challenges of RNA seq
what are some applications of RNA seq
what is CAGE and how does it work
CAGE: Cap Analysis of Gene Expression
identifies 5’ ends from stable capped RNAs at single nucleotide resolution
isolated mRNA is treated with the 5’ cap specific enzyme tobacco acid pyrophosphatase (TAP) which replaces the cap with a phosphate group, allowing the specific ligation of a 5’ adapter
a 3’ end adapter isadded (non specific) the RNA is reverse transcribed once, then subjected to PCR using the 5’ specific and 3’ non specific primers, making a cDNA library for HTS DNA sequencing
what does nuclear run on assay technique seek to investigate
how does nuclear run on assay work
- cells or tissue samples that express the gene of interest, and that the expression of that gene can be controlled by specific stimuli
- cells are chilled, lysed with sarkozyl and nuclei are isolated (this pauses RNA pol II)
- nuclei incubate at 37°C in presence of NTPs and radio labelled UTP
- new transcripts are not initiated but the UTPs become incorporated into transcripts that were being made when cells were chilled
- radio labelled RNA is isolated and hybridized to a membrane containing immobilised DNA from the gene of interest
- amount of radioactivity that hybridizes to the membrane is ~ proportional to the number of nascent transcripts
what is GRO-seq
global nuclear run on coupled with high throughput sequencing
modified run on assay that can be converted to a DNA library suitable for deep high throughput sequencing
the main alteration is using brominated nucleotides instead of labelled nucleotides
RNA molecules that have incorporated Br-UTP are purified with anti-BrdU antibodies on beads
immunoprecipitated RNA is then used to prepare compatible DNA libraries for sequencing
what were the outcomes/findings of GRO sequencing?
- allows unbiased mapping of nascent transcripts genome wide
- very sensitive and gives crucial information about RNA pol II density at different classes of genes
- revealed that RNA pol II fires bidirectionally at most mammalian promoters and enhancers and initiating ncRNAs that are transcribed antisense with respect to mRNA
- can differentiate between transcriptionally active and inactive regions of the genome
- has revealed that RNA pol II is engaged in ~30% of human genes
- transcription extends beyond mRNA 3’ cleavage site and antisense transcription is prevalent
what have high resolution methods revealed about transcription initiation
why is mapping open chromatin important
how does DNase sequencing and FAIRE sequencing work
what are the outcomes of DNase sequencing
how does chromatin accessibility profiling by ATAC-seq work
- ATAC-seq identifies accessible DNA regions by probing open chromatin with hyperactive mutant Tn5 transposase that inserts sequencing adapters into open regions of the genome
-in “tagmentation” Tn5 transposase cleaves and tags double stranded DNA with sequencing adaptors - the tagged DNA fragments are then purified, PCR amplified and sequenced
- sequencing reads can then be used to infer regions of increased accessibility as well as to map regions of transcription factor binding sites and nucleosome positions
- number of reads for a region correlates with how open that chromatin is, at single nucleotide resolution
- ATAC seq needs no sonication or phenol chloroform extraction, no antibodies and no sensitive enzymatic digestion
how does ChIP seq technology work
- used to map binding sites of TFs in the genome
- DNA binding protein is crosslinked to DNA in vivo by treating cells/tissue with formaldehyde
- chromatin is sheared by sonication into small fragments (200-600bp)
- very specific antibody against that factor of interest is used to immunoprecipitate the DNA protein complex
- crosslinks are reversed and released DNA can be amplified (if needed)
- sequence the DNA
what are the applications of ChIP seq
how does ChIP seq of RNA pol II work
what are some histone marks, their location and their significance
