Table 2.
Comparison of chromatin-associated RNA profiling methodologies: Data-based comparative analysis for the ability of these techniques to detect expression independent chromatin targeting of lncRNAs.
| ChRIP-seq (Act-D Treatment) |
PIRCh-seq | GRID-seq | MARGI-seq | ChAR-seq | ||
|---|---|---|---|---|---|---|
| Active XH lncCARs | Inactive lncCARs | |||||
| Cell lines used | BT-549 | BT-549 | H9, HFF, mESC, MEF and mNPC | MBA-MB-231, mESC, S2 | hESC, HEK | CME-W1-cl8+ |
| Organism | Human | Human | Human and Mouse | Human, Mouse and Drosophila | Human | Drosophila |
| Number of cells/chromatin required | 50–60 µg chromatin per IP | 50–60 µg chromatin per IP | 10–20 µg chromatin per IP | 5–10 µg chromatin per library | 10,000–20,000 µg chromatin per library | 100–400 million Drosophila cells per library |
| Crosslinking | 1% Formaldehyde | UV and 1% Formaldehyde | 1% Glutaraldehyde | Formaldehyde and DSG | 1% Formaldehyde | 1% Formaldehyde |
| Probes or oligos | Antibody based | Antibody based | Antibody based | Customized biotinylated bivalent linker | Ligation based: customized linker DNA. | Biotinylated oligonucleotide bridge (linker DNA) |
| Technical limitations | Chromatin fragment size | Chromatin fragment size | Chromatin fragment size | Frequency of AluI restriction sites in the genome | Specificity of linker ligation to RNA and the proximity of bound RNA to free DNA ends (fragment size) | Specificity of bridge ligation to RNA and the proximity of bound RNAs to free DNA ends (fragment size) |
| Number of chromatin bound ncRNAs | 209 | 276 | 258 | 72 (7.36%) | Not provided | Less ncRNA and abundant mRNAs |
| Overrepresented class of ncRNAs | 191 lncRNAs out of 209 ncRNAs | lncRNAs and novel transcripts (“cuffs”) | 247 lncRNAs out of 258 ncRNAs | 32 lncRNAs MALAT1, NEAT1 and U2 snRNA, roX2, snoRNAs |
Not provided | 18% snoRNA 19% snRNA |
| Relation with steady state levels of nuclear expression | Chromatin enrichment of active lncCARs independent of steady state nuclear levels | Information not provided | lncRNAs overrepresented as compared to mRNAs or other ncRNAs that generally has higher expression. | Positively correlated | Positively correlated | Positively correlated |
| Nascent transcript enrichment | Actinomycin D treated cells were used for the assay. Functionally characterized active XH lncCARs were validated for transcription independent chromatin enrichment | Not mentioned | Less compared to GRID-seq [96], CAR [57] and CPE [99] (Chromatin pellet extract) data | Nascent transcripts are enriched | In HEK cell pxRNA peaks detected in 69.1% of all the transcription start sites. DiRNA peaks detected in 61% of all the transcription start sites | Yes. Positive correlation with (Permissive nuclear Run-On sequencing) PRO-seq data [100] |
| Mechanism of action | Active XH lncCARs regulate transcription in cis (FOXD3-AS1, HOXC13-AS, GATA6-AS1 and HOXC-AS2) | One of the inactive CARs Meg3 regulates TGF-ß pathway genes in trans via triplex formation | Validated chromatin targeting of lnc-Nr2f1 | No | No | No |
| References | [28] | [33] | [95] | [96] | [97] | [98] |