Epigenetics techniques

Question 1

ATAC-seq

Answer 1

Assay for Transposase-
Accessible Chromatin

Question 2

Principle of ATAC seq

Answer 2

TN5 transposase binds to open chromatin exclusively,
in the resulting open DNA regions sequencing adaptors are inserted by the transposase. Further next-gen seq provides info on how openly accessible is the region based on the amount of copies. This freq can be plotted.

Question 3

Mechanism of ChipSeq

Answer 3

Cross-linking between DNA and proteins
(Formaldehyde)
- Cell lysis and DNA fragmentation “sonication
or enzymatic digestion”
- Antibody binding to protein of interest ->
immunoprecipitation (magnetic separation)
- Decrosslinking (heat incubation or digestion
of protein component)
- Purification (separate DNA from cellular
debris)
- Sequencing of DNA involved in binding

Question 4

Applications of ChipSeq

Answer 4

Transcription factor binding site identification ⇒
understand gene regulation/expression
- Histone modification profiling (identify
methylation, acetylation, etc.)
- Mapping of DNA-Protein interactions
- Drug discovery and development: quantify effect
of drug on specific DNA-Protein binding
- Investigation of Epigenetic dysregulation in
diseases

Question 5

3C steps

Answer 5

crosslink, digest, ligation, reverse crosslink, processing, quantification

Question 6

4C

Answer 6

like 3C but instead of two DNA regions compares one relative to the genome

Question 7

Hi-C

Answer 7

genome vs genome proximity

Question 8

Me-DIP

Answer 8

Affinity based method: Methylated DNA immunoprecipitation via anti-5mC
antibody.
- Used to study 5mC (“5th base”) or 5hmC (“6th base”) modification.
- Developed to overcome limitations of restriction enzyme (seq. specific) and
bisulfite conversion (small scale, laborious) in 2005

Question 9

Applications of Me-DIP

Answer 9

Discovery that inactive X-chromosome in females is hypermethylated on a
chromosome wide level using MeDIP coupled with microarray. [5]
- MeDIP-chip approach to investigate human breast cancer for methylation
associated silencing and observed the inactivation of the HOXA gene cluster

Question 10

What kind of methylation can Me-DIP detect

Answer 10

Can detect 5-mC methylations that
are not on the CG dipole but no mCpG.

Question 11

Me-DIP procedure

Answer 11

purify the
DNA
Sonificate
the DNA
Do the controls at the
same time to test the
antibody binding
Immunoprecipitate
with an antibody
and magnetic beads
Amplify the
result with
PCR

Question 11

MED-seq mechanism

Answer 11

Restriction enzyme recognizes methylated CpGs
2. Cuts 16 bp downstream
3. Fragments are filtered based on size
4. PCR and sequencing
5. Align to reference

Question 12

NanostringNcounter

Answer 12

uses fluorescent barcode-tagged mRNA complement. Particularly useful for low amounts of mRNA as it does not require any amplification