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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.

○ By 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 at least 100 cells per perturbation on average.

○ 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. Grouping cells based on gRNA expression: Cells are naturally grouped according to 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. In vivo, the CRISPR-Cas9 system induces various perturbations in each Cas9-expressing cell depending on the gRNA.

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) and DNA.

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

⑤ 3^rd. Use sonication to break DNA into small fragments (200-350 bp); 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 linked 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. Proceeds with PCR amplification, fragment size selection, adaptor addition.

⑨ 7^th. Compare the sequencing results to the genome reference to identify binding sites of transcription factors, etc.

Figure 3. ChIP-seq process

⑩ Application 1. ChIP-chip: HTS version of ChIP-chip is ChIP-seq.

⑪ Application 2. ChIP-PET

⑫ Application 3. ChIA-PET: ChIP-seq identifies only binding locations of specific proteins, while ChIA-PET investigates interactions between bound DNA regions.

⑬ Application 4. RIP-seq

⑭ Application 5. CLIP-seq

⑮ Application 6. meDIP-seq: DNA methylation or hydroxymethylation.

⑯ Application 7. ChIP-exo: Uses lambda exonuclease digestion. Difficult experimentally.

⑰ Application 8. CUT&RUN (Cleavage under Targets and Release using Nuclease)

○ Uses Micrococcal Nuclease (MNase).

⑱ Application 9. CUT&Tag (Cleavage under Targets and Tagmentation)

○ Overview

○ Tn5 transposase is an enzyme that cuts open regions of DNA, generating various fragments such as single nucleosomes, dimers, trimers, and more.

○ The Tn5 transposase catalyzes DNA insertion by creating sticky ends.

○ It can directly merge adaptors, allowing immediate sequencing.

○ Procedure

○ Step 1. Bind nuclei to beads to anchor them. Magnetic capture leads to high retention of cells.

○ Step 2. Add primary antibody: Quality of data is highly dependent on quality (specificity & sensitivity) of antibody.

○ Step 3. Add secondary antibody.

○ Step 4. Conjugated Tn5-transposase binds to complex and cuts surrounding areas of open DNA.

○ Step 5. PCR amplification, fragment size selection, sequencing

○ Types

○ MuLTI-Tag: Minimizes crossover in multiplexing via direct barcode conjugation.

○ multi-CUT&Tag: Uses barcoded Tn5/pA-antibody complexes. Identifies colocalization of marks.

○ spatial-CUT&Tag: Spatially visualizes histone modifications and chromatin states on tissue sections.

Table 1. CUT&Tag vs. CUT&RUN vs. ChIP-Seq (ref, ref, ref, ref)

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 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.

○ Tn5 transposase is an enzyme that cuts open regions of DNA, generating various fragments such as single nucleosomes, dimers, trimers, and more.

○ The Tn5 transposase catalyzes DNA insertion by creating sticky ends.

○ It can directly merge adaptors, allowing immediate sequencing.

○ The ~10 bp periodicity observed in ATAC-seq is related to the fact that one full turn of the DNA helix requires 10 bp, which is associated with the minor groove distance.

Figure 5. ATAC-seq fragment size distribution

② Type 1. bulk ATAC-seq

○ Step 1. Nuclei isolation

○ Step 2. Tn5 transposase treatment: This cuts less condensed open regions in the chromosome 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

⑤ Type 4. FAIRE-seq: Formaldehyde to crosslink chromatin, and phenol-chloroform to extract sheared DNA.

⑥ Type 5. DNaseI-seq: DNase I endonuclease to digest chromatin. Can identify the types of transcription factors interacting with chromatin.

Figure 6. Example results of DNAseI-seq

⑦ Type 6. MNase-seq: Endo-exonuclease to processively digest DNA until obstruction.

Figure 7. Comparison of ATAC-seq with Various Sequencing Techniques

⑺ NOMe-seq (nucleosome occupancy and methylome sequencing)

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

② Identifies transcription factors interacting with chromatin.

⑻ MBD-seq

⑼ Bru-seq & BruChase-seq

① Newly transcribed nascent RNA is labeled with Bru (bromouridine) and then sequenced.

② Used for studying RNA synthesis, RNA stability, and splicing.

⑽ TT-seq

⑾ MNIST-seq: RNA methylation on chromatin regulation

⑿ GRO-seq: global run-on sequencing

3. Type 3. Identifying post-translational regulation

⑴ Ribo-seq

① Sequencing of ribosome-protected RNA, indicating active translation.

② scRibo-seq

○ Measures ribosomal occupancy per single codon.

○ 1^st. FACS and lysis.

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

○ 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 at the site where APOBEC and mRNA bind, replacing cytosine (C) with uracil (U).

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. When the target transcript is present, it forms a dsRNA structure, allowing ADAR to induce A-to-C editing.

② 2^nd. This editing induces cellular behaviors such as GFP expression and caspase activation.

⑵ LADL (light-activated dynamic looping)

① An example of photo-activatable gene expression.

Input: 2022.01.10 00:03

Modified: 2023.01.28 23:12