PMID- 35379945
OWN - NLM
STAT- MEDLINE
DCOM- 20220512
LR  - 20230823
IS  - 1750-2799 (Electronic)
IS  - 1750-2799 (Linking)
VI  - 17
IP  - 5
DP  - 2022 May
TI  - RASER-FISH: non-denaturing fluorescence in situ hybridization for preservation of 
      three-dimensional interphase chromatin structure.
PG  - 1306-1331
LID - 10.1038/s41596-022-00685-8 [doi]
AB  - DNA fluorescence in situ hybridization (FISH) has been a central technique in 
      advancing our understanding of how chromatin is organized within the nucleus. 
      With the increasing resolution offered by super-resolution microscopy, the 
      optimal maintenance of chromatin structure within the nucleus is essential for 
      accuracy in measurements and interpretation of data. However, standard 3D-FISH 
      requires potentially destructive heat denaturation in the presence of chaotropic 
      agents such as formamide to allow access to the DNA strands for labeled FISH 
      probes. To avoid the need to heat-denature, we developed Resolution After 
      Single-strand Exonuclease Resection (RASER)-FISH, which uses exonuclease 
      digestion to generate single-stranded target DNA for efficient probe binding over 
      a 2 d process. Furthermore, RASER-FISH is easily combined with immunostaining of 
      nuclear proteins or the detection of RNAs. Here, we provide detailed procedures 
      for RASER-FISH in mammalian cultured cells to detect single loci, chromatin 
      tracks and topologically associating domains with conventional and 
      super-resolution 3D structured illumination microscopy. Moreover, we provide a 
      validation and characterization of our method, demonstrating excellent 
      preservation of chromatin structure and nuclear integrity, together with improved 
      hybridization efficiency, compared with classic 3D-FISH protocols.
CI  - (c) 2022. The Author(s), under exclusive licence to Springer Nature Limited.
FAU - Brown, Jill M
AU  - Brown JM
AUID- ORCID: 0000-0002-8884-2047
AD  - MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, 
      Oxford University, Oxford, UK.
FAU - De Ornellas, Sara
AU  - De Ornellas S
AD  - MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, 
      Oxford University, Oxford, UK.
FAU - Parisi, Eva
AU  - Parisi E
AUID- ORCID: 0000-0001-7567-9845
AD  - MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, 
      Oxford University, Oxford, UK.
AD  - School of Clinical Medicine, Addenbrooke's Hospital, Cambridge, UK.
FAU - Schermelleh, Lothar
AU  - Schermelleh L
AUID- ORCID: 0000-0002-1612-9699
AD  - Micron Oxford Advanced Bioimaging Unit, Department of Biochemistry, Oxford 
      University, Oxford, UK.
FAU - Buckle, Veronica J
AU  - Buckle VJ
AUID- ORCID: 0000-0003-1195-2608
AD  - MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, 
      Oxford University, Oxford, UK. veronica.buckle@imm.ox.ac.uk.
LA  - eng
SI  - figshare/10.6084/m9.figshare.16778899
SI  - figshare/10.6084/m9.figshare.16778902
SI  - figshare/10.6084/m9.figshare.16755394
GR  - MC_UU_00016/1/MRC_/Medical Research Council/United Kingdom
GR  - MR/N00969X/1/MRC_/Medical Research Council/United Kingdom
GR  - MR/K015777X/1/MRC_/Medical Research Council/United Kingdom
GR  - BB/L01811X/BB_/Biotechnology and Biological Sciences Research Council/United 
      Kingdom
GR  - 104924/Z/14/Z/WT_/Wellcome Trust/United Kingdom
GR  - 091911/WT_/Wellcome Trust/United Kingdom
GR  - 107457/Z/15/Z/WT_/Wellcome Trust/United Kingdom
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Review
DEP - 20220404
PL  - England
TA  - Nat Protoc
JT  - Nature protocols
JID - 101284307
RN  - 0 (Chromatin)
RN  - 9007-49-2 (DNA)
RN  - EC 3.1.- (Exonucleases)
SB  - IM
MH  - Animals
MH  - *Cell Nucleus/genetics/metabolism
MH  - *Chromatin/metabolism
MH  - DNA/genetics/metabolism
MH  - Exonucleases/metabolism
MH  - In Situ Hybridization, Fluorescence/methods
MH  - Interphase
MH  - Mammals
EDAT- 2022/04/06 06:00
MHDA- 2022/05/14 06:00
CRDT- 2022/04/05 05:32
PHST- 2021/08/03 00:00 [received]
PHST- 2022/01/10 00:00 [accepted]
PHST- 2022/04/06 06:00 [pubmed]
PHST- 2022/05/14 06:00 [medline]
PHST- 2022/04/05 05:32 [entrez]
AID - 10.1038/s41596-022-00685-8 [pii]
AID - 10.1038/s41596-022-00685-8 [doi]
PST - ppublish
SO  - Nat Protoc. 2022 May;17(5):1306-1331. doi: 10.1038/s41596-022-00685-8. Epub 2022 
      Apr 4.