13. We recommend fluorescent eye markers (e.g., RFP or GFP).
These are most clearly identified in a white (w) mutant background. If a w + background is used, adult flies should be
screened 3 days after emergence for fluorescence. Ommatidia
near the equator are the most conspicuous spots of fluorescent
signals.
14. Adjust dorsal-ventral orientation using the dissecting needle.
Gently rotate the embryos on the needle tip with the help of
the broken chorion.
References
1. Furlong EEM, Levine M (2018) Developmental enhancers and chromosome topology. Science 361(6409):1341–1345. https://doi.org/
10.1126/science.aau0320
2. Dekker J, Belmont AS, Guttman M, Leshyk
VO, Lis JT, Lomvardas S, Mirny LA, O’Shea
CC, Park PJ, Ren B, Politz JCR, Shendure J,
Zhong S, Network DN (2017) The 4D
nucleome
project.
Nature
549
(7671):219–226. https://doi.org/10.1038/
nature23884
3. Vermunt MW, Zhang D, Blobel GA (2019)
The interdependence of gene-regulatory elements and the 3D genome. J Cell Biol 218
(1):12–26.
https://doi.org/10.1083/jcb.
201809040
4. Beagrie RA, Scialdone A, Schueler M, Kraemer
DC, Chotalia M, Xie SQ, Barbieri M, de
Santiago I, Lavitas LM, Branco MR, Fraser J,
Dostie J, Game L, Dillon N, Edwards PA,
Nicodemi M, Pombo A (2017) Complex
multi-enhancer contacts captured by genome
architecture
mapping.
Nature
543
(7646):519–524. https://doi.org/10.1038/
nature21411
5. Gibcus JH, Samejima K, Goloborodko A,
Samejima I, Naumova N, Nuebler J, Kanemaki
MT, Xie L, Paulson JR, Earnshaw WC, Mirny
LA, Dekker J (2018) A pathway for mitotic
chromosome formation. Science 359(6376):
eaaao6135.
https://doi.org/10.1126/sci
ence.aao6135
6. Larson DR, Zenklusen D, Wu B, Chao JA,
Singer RH (2011) Real-time observation of
transcription initiation and elongation on an
endogenous yeast gene. Science 332
(6028):475–478. https://doi.org/10.1126/
science.1202142
7. Hocine S, Raymond P, Zenklusen D, Chao JA,
Singer RH (2013) Single-molecule analysis of
gene expression using two-color RNA labeling
in live yeast. Nat Methods 10(2):119–121.
https://doi.org/10.1038/nmeth.2305
8. Golding I, Paulsson J, Zawilski SM, Cox EC
(2005) Real-time kinetics of gene activity in
individual bacteria. Cell 123(6):1025–1036.
https://doi.org/10.1016/j.cell.2005.09.031
9. Garcia HG, Tikhonov M, Lin A, Gregor T
(2013) Quantitative imaging of transcription
in living Drosophila embryos links polymerase
activity to patterning. Curr Biol 23
(21):2140–2145. https://doi.org/10.1016/j.
cub.2013.08.054
10. Fukaya T, Lim B, Levine M (2016) Enhancer
control of transcriptional bursting. Cell 166
(2):358–368. https://doi.org/10.1016/j.cell.
2016.05.025
11. Lucas T, Ferraro T, Roelens B, De Las Heras
Chanes J, Walczak AM, Coppey M, Dostatni N
(2013) Live imaging of bicoid-dependent transcription in Drosophila embryos. Curr Biol 23
(21):2135–2139. https://doi.org/10.1016/j.
cub.2013.08.053
12. Saad H, Gallardo F, Dalvai M, Tanguy-leGac N, Lane D, Bystricky K (2014) DNA
dynamics during early double-strand break
processing revealed by non-intrusive imaging
of living cells. PLoS Genet 10(3):e1004187.
https://doi.org/10.1371/journal.pgen.
1004187
13. Mariame B, Kappler-Gratias S, Kappler M,
Balor S, Gallardo F, Bystricky K (2018) Realtime visualization and quantification of human
cytomegalovirus replication in living cells using
the ANCHOR DNA labeling technology.
J Virol 92(18):e00571–e00518. https://doi.
org/10.1128/JVI.00571-18
14. Dubarry N, Pasta F, Lane D (2006) ParABS
systems of the four replicons of Burkholderia
cenocepacia: new chromosome centromeres
confer partition specificity. J Bacteriol 188
(4):1489–1496. https://doi.org/10.1128/
JB.188.4.1489-1496.2006
15. Gratz SJ, Harrison MM, Wildonger J, O’Connor-Giles KM (2015) Precise genome editing
of Drosophila with CRISPR RNA-guided
Imaging of Chromatin Loci and Transcription
383
These are most clearly identified in a white (w) mutant background. If a w + background is used, adult flies should be
screened 3 days after emergence for fluorescence. Ommatidia
near the equator are the most conspicuous spots of fluorescent
signals.
14. Adjust dorsal-ventral orientation using the dissecting needle.
Gently rotate the embryos on the needle tip with the help of
the broken chorion.
References
1. Furlong EEM, Levine M (2018) Developmental enhancers and chromosome topology. Science 361(6409):1341–1345. https://doi.org/
10.1126/science.aau0320
2. Dekker J, Belmont AS, Guttman M, Leshyk
VO, Lis JT, Lomvardas S, Mirny LA, O’Shea
CC, Park PJ, Ren B, Politz JCR, Shendure J,
Zhong S, Network DN (2017) The 4D
nucleome
project.
Nature
549
(7671):219–226. https://doi.org/10.1038/
nature23884
3. Vermunt MW, Zhang D, Blobel GA (2019)
The interdependence of gene-regulatory elements and the 3D genome. J Cell Biol 218
(1):12–26.
https://doi.org/10.1083/jcb.
201809040
4. Beagrie RA, Scialdone A, Schueler M, Kraemer
DC, Chotalia M, Xie SQ, Barbieri M, de
Santiago I, Lavitas LM, Branco MR, Fraser J,
Dostie J, Game L, Dillon N, Edwards PA,
Nicodemi M, Pombo A (2017) Complex
multi-enhancer contacts captured by genome
architecture
mapping.
Nature
543
(7646):519–524. https://doi.org/10.1038/
nature21411
5. Gibcus JH, Samejima K, Goloborodko A,
Samejima I, Naumova N, Nuebler J, Kanemaki
MT, Xie L, Paulson JR, Earnshaw WC, Mirny
LA, Dekker J (2018) A pathway for mitotic
chromosome formation. Science 359(6376):
eaaao6135.
https://doi.org/10.1126/sci
ence.aao6135
6. Larson DR, Zenklusen D, Wu B, Chao JA,
Singer RH (2011) Real-time observation of
transcription initiation and elongation on an
endogenous yeast gene. Science 332
(6028):475–478. https://doi.org/10.1126/
science.1202142
7. Hocine S, Raymond P, Zenklusen D, Chao JA,
Singer RH (2013) Single-molecule analysis of
gene expression using two-color RNA labeling
in live yeast. Nat Methods 10(2):119–121.
https://doi.org/10.1038/nmeth.2305
8. Golding I, Paulsson J, Zawilski SM, Cox EC
(2005) Real-time kinetics of gene activity in
individual bacteria. Cell 123(6):1025–1036.
https://doi.org/10.1016/j.cell.2005.09.031
9. Garcia HG, Tikhonov M, Lin A, Gregor T
(2013) Quantitative imaging of transcription
in living Drosophila embryos links polymerase
activity to patterning. Curr Biol 23
(21):2140–2145. https://doi.org/10.1016/j.
cub.2013.08.054
10. Fukaya T, Lim B, Levine M (2016) Enhancer
control of transcriptional bursting. Cell 166
(2):358–368. https://doi.org/10.1016/j.cell.
2016.05.025
11. Lucas T, Ferraro T, Roelens B, De Las Heras
Chanes J, Walczak AM, Coppey M, Dostatni N
(2013) Live imaging of bicoid-dependent transcription in Drosophila embryos. Curr Biol 23
(21):2135–2139. https://doi.org/10.1016/j.
cub.2013.08.053
12. Saad H, Gallardo F, Dalvai M, Tanguy-leGac N, Lane D, Bystricky K (2014) DNA
dynamics during early double-strand break
processing revealed by non-intrusive imaging
of living cells. PLoS Genet 10(3):e1004187.
https://doi.org/10.1371/journal.pgen.
1004187
13. Mariame B, Kappler-Gratias S, Kappler M,
Balor S, Gallardo F, Bystricky K (2018) Realtime visualization and quantification of human
cytomegalovirus replication in living cells using
the ANCHOR DNA labeling technology.
J Virol 92(18):e00571–e00518. https://doi.
org/10.1128/JVI.00571-18
14. Dubarry N, Pasta F, Lane D (2006) ParABS
systems of the four replicons of Burkholderia
cenocepacia: new chromosome centromeres
confer partition specificity. J Bacteriol 188
(4):1489–1496. https://doi.org/10.1128/
JB.188.4.1489-1496.2006
15. Gratz SJ, Harrison MM, Wildonger J, O’Connor-Giles KM (2015) Precise genome editing
of Drosophila with CRISPR RNA-guided
Imaging of Chromatin Loci and Transcription
383