Transcriptomics Flashcards
How do we measure how much a gene is expressed?
What is PCR
How is qPCR done with a reporter probe
How does qPCR work
What is qPCR normalisation
Use the total amount of RNA or DNA in each sample as a normalization factor.
Quantify RNA/DNA input before reverse transcription or amplification and adjust qPCR data accordingly.
What are the limitations of qPCR
What are microarrays
How does an Affy Gene chip work*
An Affymetrix GeneChip (or Affy GeneChip) is a microarray technology used to measure gene expression or detect variations in DNA, such as SNPs
A glass or silicon chip is embedded with thousands to millions of unique oligonucleotide probes.
Each probe is complementary to a specific sequence of the target RNA or DNA.
Perfect Match (PM) Probes: Complementary to the target sequence.
Mismatch (MM) Probes: Have a single base mismatch to account for nonspecific hybridization.
Total RNA or mRNA is extracted from the sample and reverse transcibed into cDNA
e labeled cRNA is fragmented to ensure optimal binding and hybridized onto the chip.
The cRNA binds to complementary probe sequences on the array through base-pairing rules.
The chip is scanned using a laser scanner to measure fluorescence intensity at each probe location.
Fluorescence Intensity: Proportional to the amount of hybridized RNA, which reflects the expression level of the corresponding gene.
What are the limitations of using a microarray
How does next generation sequencing work
What can RNA -seq be used to do
Give an overview of rna-seq
How is rRNA removed in RNA-seq
How is RNA prepared for sequencing
What is RNA-seq normalisation and how is it done
What is single cell RNA-seq + problems with RNA-seq
What is the Single Cell RNA-seq Workflow
How is Single Cell RNA-seq Data Analysed*
Build a graph of cell-cell similarities (e.g., k-nearest neighbors) and identify clusters
How can data from Single Cell RNA-seq be plotted
What methods can be used for profiling binding of transcription factors and histone modifications
What can CHIP sequencing be used to do
Give the Chip-seq Workflow
How is the data visualised from Chip-seq analysis*
Peaks representing regions of DNA enriched for the protein of interest are seen
What are the limitations of Chip-seq
How does cut and run work*
The chromatin is maintained in its native state without crosslinking
A specific antibody targeting the protein of interest (or histone modification) is added.
The antibody binds to its target protein on the chromatin.
A fusion of protein A or protein G (which binds to the antibody) and micrococcal nuclease (MNase) is introduced.
The MNase is tethered to the chromatin via the antibody-protein interaction
The nuclease selectively cleaves DNA near the bound protein-antibody complex.
Cleaved DNA fragments are released into the supernatant, while other chromatin remains intact.
The released DNA fragments are purified.
These fragments correspond to regions of the genome where the protein of interest was bound
The isolated DNA is prepared for sequencing
What are the methods for identification of open chromatin regions
What are DNA HS sites
Give an overview of DNase-seq
What is ATAC-seq + advantages
What does Tn5 do in ATAC-seq
How can Profiling of chromatin accessibility be done with ATAC-seq ( how is data obtained interpreted)
How does MNase-seq work
What Methods are used to map binding of RNA-binding proteins
How does RIP-seq and CLIP-seq work
Why is knowledge about chromatin interactions useful
How can ligations that occur between different ChiP complexes be identified*
Crosslink chromatin to preserve protein-DNA interactions.
Fragment DNA and perform proximity ligation to join spatially close DNA fragments.
Perform immunoprecipitation using specific antibodies for the proteins of interest.
Sequence and analyze paired-end reads to identify ligation events.
What does the ENCODE project aim to do
what are ChIP complexes
ChIP complexes refer to the molecular assemblies that are targeted and analyzed during Chromatin Immunoprecipitation (ChIP) experiments. These complexes typically include proteins bound to specific regions of DNA and any associated cofactors or regulatory elements.
