PMID- 38529487
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20240401
DP  - 2024 Feb 19
TI  - Allele-level visualization of transcription and chromatin by high-throughput 
      imaging.
LID - 2024.02.19.580973 [pii]
LID - 10.1101/2024.02.19.580973 [doi]
AB  - The spatial arrangement of the genome within the nucleus is a pivotal aspect of 
      cellular organization and function with implications for gene expression and 
      regulation. While all genome organization features, such as loops, domains, and 
      radial positioning, are non-random, they are characterized by a high degree of 
      single-cell variability. Imaging approaches are ideally suited to visualize, 
      measure, and study single-cell heterogeneity in genome organization. Here, we 
      describe two methods for the detection of DNA and RNA of individual gene alleles 
      by fluorescence in situ hybridization (FISH) in a high-throughput format. We have 
      optimized combined DNA/RNA FISH approaches either using simultaneous or 
      sequential detection. These optimized DNA and RNA FISH protocols, implemented in 
      a 384-well plate format alongside automated image and data analysis, enable 
      accurate detection of chromatin loci and their gene expression status across a 
      large cell population with allele-level resolution. We successfully visualized 
      MYC and EGFR DNA and RNA in multiple cell types, and we determined the radial 
      position of active and inactive MYC and EGFR alleles. These optimized DNA/RNA 
      detection approaches are versatile and sensitive tools for mapping of chromatin 
      features and gene activity at the single-allele level and at high throughput.
FAU - Almansour, Faisal
AU  - Almansour F
AD  - Cell Biology of Genomes, National Cancer Institute, National Institute of Health, 
      Bethesda, MD 20892, USA.
AD  - Department of Biochemistry and Molecular and Cellular Biology, Georgetown 
      University Medical School, Washington, DC, 20057, USA.
FAU - Keikhosravi, Adib
AU  - Keikhosravi A
AD  - High-Throughput Imaging Facility, National Cancer Institute, National Institute 
      of Health, Bethesda, MD 20892, USA.
FAU - Pegoraro, Gianluca
AU  - Pegoraro G
AUID- ORCID: 0000-0003-2843-9464
AD  - High-Throughput Imaging Facility, National Cancer Institute, National Institute 
      of Health, Bethesda, MD 20892, USA.
FAU - Misteli, Tom
AU  - Misteli T
AUID- ORCID: 0000-0003-3530-3020
AD  - Cell Biology of Genomes, National Cancer Institute, National Institute of Health, 
      Bethesda, MD 20892, USA.
LA  - eng
GR  - Z01 BC010309/ImNIH/Intramural NIH HHS/United States
PT  - Preprint
DEP - 20240219
PL  - United States
TA  - bioRxiv
JT  - bioRxiv : the preprint server for biology
JID - 101680187
PMC - PMC10962702
OTO - NOTNLM
OT  - DNA/RNA FISH
OT  - Genome organization
OT  - High-throughput imaging
OT  - In Situ Hybridization
COIS- Conflict of Interest The authors declare no conflicts of interest.
EDAT- 2024/03/26 06:45
MHDA- 2024/03/26 06:46
PMCR- 2024/03/25
CRDT- 2024/03/26 03:46
PHST- 2024/03/26 06:45 [pubmed]
PHST- 2024/03/26 06:46 [medline]
PHST- 2024/03/26 03:46 [entrez]
PHST- 2024/03/25 00:00 [pmc-release]
AID - 2024.02.19.580973 [pii]
AID - 10.1101/2024.02.19.580973 [doi]
PST - epublish
SO  - bioRxiv [Preprint]. 2024 Feb 19:2024.02.19.580973. doi: 
      10.1101/2024.02.19.580973.