EpigenomicsFinalFC1 - Assays & PTMs
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What is the general scheme shared by most epigenomics techniques?
Enrich DNA based on a chromatin property then compare test vs input using high-throughput sequencing to identify genome-wide enrichment patterns
Why is input DNA essential in epigenomics experiments?
It controls for fragmentation bias sequence bias and copy number so enrichment reflects true chromatin features
Why did epigenomics require high-throughput sequencing?
Because chromatin features require genome-wide high-resolution quantitative measurement not possible with single-locus methods
What does DNase-seq measure?
Chromatin accessibility and nucleosome-depleted regions
How does DNase-seq work?
DNase I preferentially cuts accessible DNA and sequencing of cut sites reveals open chromatin regions
What are DNase I hypersensitive sites (DHSs)?
Short genomic regions with high DNase cleavage corresponding to promoters enhancers and transcription factor binding sites
How can DNase-seq identify transcription factor binding sites?
Bound transcription factors protect DNA locally creating a footprint within a broader DNase hypersensitive region
What is ATAC-seq?
A chromatin accessibility assay using a transposase to insert sequencing adapters into open chromatin
Why is ATAC-seq considered time-saving?
Fragmentation and adapter ligation occur in a single step with low input requirements
What extra information can ATAC-seq provide beyond accessibility?
Nucleosome positioning and phasing information
What does MNase-seq measure?
Genome-wide nucleosome positioning
How does MNase-seq work?
MNase digests linker DNA leaving nucleosome-protected fragments that are sequenced
What does a well-positioned nucleosome look like in MNase-seq data?
A sharp reproducible peak at a defined genomic location
What nucleosome pattern is typical of actively transcribed genes?
A nucleosome-depleted promoter a strong +1 nucleosome and phased nucleosomes across the gene body
What does ChIP-seq measure?
Genomic localization of histone modifications or DNA-binding proteins
What are the core steps of ChIP-seq?
Crosslink chromatin shear DNA immunoprecipitate with antibody purify DNA and sequence
What determines the quality of a ChIP-seq experiment?
Antibody specificity and affinity
Why can sonication bias be a problem in ChIP-seq?
Heterochromatin is harder to shear leading to under-representation if not controlled
How do CUT&RUN and CUT&Tag improve on ChIP-seq?
They cleave DNA in situ at antibody-bound sites producing higher resolution lower background and requiring fewer cells
What is the key conceptual difference between ChIP-seq and CUT&RUN?
ChIP pulls down chromatin fragments while CUT&RUN releases only local fragments at binding sites
What does meDIP-seq detect?
Regions enriched for methylated cytosines (5mC)
What is the difference between meDIP and MeCP-affinity methods?
meDIP uses antibodies while MeCP uses methyl-CpG binding proteins but both give regional not base-resolution data
What is the principle of bisulfite sequencing?
Unmethylated cytosines are converted to uracil while methylated cytosines remain cytosine
What unique information does bisulfite-seq provide?
Single-base resolution DNA methylation status
What is a limitation of standard bisulfite sequencing?
It cannot distinguish 5-methylcytosine from 5-hydroxymethylcytosine
What do 3C-based techniques measure?
Physical proximity of genomic regions in three-dimensional nuclear space
What are the core steps of 3C or Hi-C?
Crosslink chromatin digest DNA ligate nearby fragments and sequence ligation products
What does a Hi-C interaction matrix represent?
Contact frequency between all pairs of genomic loci
What genomic structures are revealed by Hi-C?
Chromatin loops TADs and A/B compartments
3C plus high-throughput sequencing detects what?
Long-range chromatin interactions
MNase plus high-throughput sequencing detects what?
Nucleosome positioning
ChIP plus high-throughput sequencing detects what?
Protein or histone modification localization
Bisulfite plus high-throughput sequencing detects what?
Base-resolution DNA methylation
MeCP-affinity plus high-throughput sequencing detects what?
Regions enriched for methylated DNA
In epigenomics data what does a peak usually indicate?
Enrichment of the assayed chromatin feature at that locus
In accessibility assays what does a depletion indicate?
Nucleosome occupancy or repressive chromatin
Note 
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