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Chapter 10-2. Epigenomics Sequencing

Recommended Reading: 【Biology】 Chapter 10. Genome Projects and Sequencing Technologies

1. Type 1. Identifying gene function

2. Type 2. Identifying transcription regulation

3. Type 3. Identifying post-translational regulation

4. Type 4. Programmable cell function

a. Epigenetics Library

1. Type 1. Identifying gene function

⑴ Perturb-seq

① 1^st. Treat Cas9-expressing cells with various types of gRNA libraries

○ Using CRISPR-Cas9, various perturbations occur in each cell depending on the type of gRNA

○ At least 2.5 million cells should remain after filtering

○ There should be an average of at least 100 cells per perturbation

○ Each cell should have approximately 10,000 UMIs

② 2^nd. Use sequencing technologies that detect both gRNA and mRNA (e.g., scRNA-seq, MERFISH)

③ 3^rd. Group cells by gRNA expression: Naturally groups cells by perturbation conditions

④ 4^th. Identify gene function

○ Premise: Genes with similar functions are likely to exhibit similar expression patterns

○ Discoverable Result 1. Changes in gene expression due to perturbations

○ Discoverable Result 2. Differences in perturbation effects due to genetic variants

Figure 1. General schematic of Perturb-seq including validation experiments

⑵ in vivo Perturb-seq

① 1^st. Deliver AAV (adeno-associated virus) containing Cre to mice to induce Cas9 expression

② 2^nd. Deliver lentivirus containing sgRNA

③ 3^rd. Various perturbations occur in vivo in each Cas9-expressing cell due to gRNA via the CRISPR-Cas9 system

④ 4^th. Perform scRNA-seq and MERFISH after approximately 10 days

Figure 2. in vivo Perturb-seq process

2. Type 2. Identifying transcription regulation

⑴ BS-seq (bisulfite sequencing)

① Treat with bisulfite to determine methylation patterns

② Enables epigenomic profiling

⑵ ChIP-seq (chromatin immunoprecipitation sequencing)

① Definition: Enables analysis of DNA regions bound to specific proteins, such as transcription factors.

② Combines DNA sequencing with ChIP (chromatin immunoprecipitation) to identify binding sites of DNA-associated proteins.

③ 1^st. Treat with a cross-linking agent (e.g., formaldehyde) to fix proteins (e.g., transcription factors) to DNA.

④ 2^nd. Lyse the cells, leaving only the DNA/protein complexes.

⑤ 3^rd. Use sonication to break DNA into small fragments; cross-linked DNA remains intact.

⑥ 4^th. Add antibodies attached to magnetic beads; applying a magnetic field allows isolation of specific proteins and their associated DNA.

⑦ 5^th. Reverse cross-linking: Apply heat to the precipitate to separate DNA from proteins.

⑧ 6^th. Perform sequencing to determine the DNA sequence.

⑨ 7^th. Compare the sequencing results to the genome reference to identify binding sites of transcription factors and other proteins.

Figure 3. ChIP-seq process

⑧ Application 1. ChIP-chip

⑨ Application 2. ChIP-PET

⑩ Application 3. ChIA-PET

○ Difference: ChIP-seq identifies only binding locations of specific proteins, while ChIA-PET investigates interactions between bound DNA regions

⑶ Hi-C seq (high throughput chromatin conformation capture sequencing)

① Definition: Investigates sequences that are naturally close together on chromosomes

② Examines DNA distances to reveal the 3D folding structure of chromosomes within the nucleus

Figure 4. Hi-C seq process

③ Application 1. ChIA-PET

○ Difference: Hi-C investigates all naturally occurring DNA-DNA interactions, while ChIA-PET focuses on DNA-DNA interactions mediated by specific proteins

⑷ DNA ticker tape (prime editing)

① 1^st. Initially, only the first site is activated

② 2^nd. After the first event, the second site becomes activated

③ 3^rd. Sequentially records molecular events over time

⑸ ENGRAM (enhancer-driven genomic recording of transcriptional activity in multiplex)

① Uses perRNA linked to synthetic enhancers

② Records the sequence and intensity of signaling

③ Reference: Chen et al., bioRxiv (2021)

⑹ Tag

① MuLTI-Tag

○ Minimizes crossover in multiplexing via direct barcode conjugation

② multi-CUT&Tag

○ Uses barcoded Tn5/pA-antibody complexes

○ Identifies colocalization of marks

③ spatial-CUT&Tag

○ Spatially visualizes histone modifications and chromatin states on tissue sections

⑺ ATAC-seq (assay for transposase-accessible chromatin with sequencing)

① Overview

○ Definition: A sequencing technique that identifies euchromatin regions

○ Pseudo-expression: Euchromatin regions can be inferred as areas of gene expression

② Type 1. bulk ATAC-seq

○ Step 1. Nuclei isolation

○ Step 2. Treat with Tn5 transposase: Cuts open regions in chromatin and inserts DNA sequence tags

○ Step 3. Amplification & sequencing

○ Step 4. Data analysis

③ Type 2. scATAC-seq

○ Defines cell type-specific CREs to identify regulatory TFs and cell types associated with diseases and traits

○ Used in interpreting GWAS variants

○ Comparison of scRNA-seq and scATAC-seq

○ scRNA-seq: x_ij ∈ ℤ_≥0

○ scATAC-seq: x_ij ∈ {0, 1}, j ≫ i

④ Type 3. spatial ATAC-seq

⑻ NOMe-seq (nucleosome occupancy and methylome sequencing)

① Defines nucleosome-depleted regions (NDR) where TFs bind

② Identifies transcription factors interacting with chromatin

⑼ DNaseI-seq

① Identifies transcription factors interacting with chromatin

⑽ MNase-seq

⑾ FAIRE-seq

⑿ MBD-seq

3. Type 3. Identifying post-translational regulation

⑴ scRibo-seq

① Measures ribosomal occupancy per single codon

② 1^st. FACS and lysis

③ 2^nd. Nuclease footprinting: MNase nuclease → inactivation → release of footprints

④ 3^rd. Create small-RNA library: End repair → 3’ ligation → 5’ ligation → cDNA synthesis → indexing PCR

⑵ STAMP-RBP

① Uses scRNA-seq to identify RNA-binding proteins (RBPs)

② 1^st. Attach APOBEC to RBPs

③ 2^nd. Enable C-U editing where APOBEC and mRNA bind: Substitutes C base with U base

④ 3^rd. RNA-seq

⑤ 4^th. Use SAILOR to identify C-U editing sites

⑥ Can also identify isoform-specific binding profiles using long-read sequencing

4. Type 4. Programmable cell function

⑴ RADARS

① 1^st. Forms dsRNA with the target transcript to induce A-C editing by ADAR

② 2^nd. Editing triggers cellular behaviors like GFP or caspase activation

⑵ LADL (light-activated dynamic looping)

① Example of photo-activatable gene expression

Input: 2022.01.10 00:03

Modified: 2023.01.28 23:12