5. Our previous studies have shown that MBs with such modifications are highly resistant to nuclease degradation and biocompatible in living cells [32]. Other MBs with similar features can
potentially be used.
6. MTS-targeting MBs used in our experiments are not complementary to endogenous RNA targets in mammalian cells, and
therefore the MB fluorescence should be significantly
quenched unless hybridized to their MTS. MBs with other
sequences not complementary to endogenous RNA targets in
mammalian cells can potentially be used.
7. MBs with other dye-quencher pairs can also be used.
8. We have only tested this technique in mammalian cell lines, but
we suspect that it would be applicable in other types of cells
such as insect or plant cells.
9. Microporation was performed using equipment and buffers
included in the Thermo Fisher Neon transfection system.
Other electroporation methods can potentially be used for
MB delivery.
10. Antibiotic-free medium was used to maximize cell survival
during and after microporation.
11. Telomere-targeting FISH probes labeled with other dyes that
are optically distinct from MTSa-targeting MBs can potentially
be used.
12. We used anti-centromere antibodies derived from the serum of
human patients with calcinosis, Raynaud’s phenomenon,
esophageal dysmotility, sclerodactyly, and telangiectasia
(CREST) syndrome and Alexa647-labeled anti-human secondary antibodies for IF. Other anti-centromere antibodies
and secondary antibodies labeled with other dyes that are
optically distinct from MTSb-targeting MBs can potentially
be used.
13. We used DV2-cube (ET525/50 m, 585dcxr, ET655lp, Photometrics) for simultaneous dual-color imaging of telomeres and
centromeres.
14. Other software can potentially be used.
15. HEK293 cells attach and spread more rapidly on fibronectincoated glass surfaces.
16. FuGENE
® 6 was used for transfection in our experiments.
Other transfection regents can potentially be used.
17. Gentle pipetting minimizes cell damage.
18. When low-retention microcentrifuge tubes are used, cell pellets may not form.
19. Microporation parameters are cell line dependent.
Visualizing Genomic Loci by CRISPR/MB
369
potentially be used.
6. MTS-targeting MBs used in our experiments are not complementary to endogenous RNA targets in mammalian cells, and
therefore the MB fluorescence should be significantly
quenched unless hybridized to their MTS. MBs with other
sequences not complementary to endogenous RNA targets in
mammalian cells can potentially be used.
7. MBs with other dye-quencher pairs can also be used.
8. We have only tested this technique in mammalian cell lines, but
we suspect that it would be applicable in other types of cells
such as insect or plant cells.
9. Microporation was performed using equipment and buffers
included in the Thermo Fisher Neon transfection system.
Other electroporation methods can potentially be used for
MB delivery.
10. Antibiotic-free medium was used to maximize cell survival
during and after microporation.
11. Telomere-targeting FISH probes labeled with other dyes that
are optically distinct from MTSa-targeting MBs can potentially
be used.
12. We used anti-centromere antibodies derived from the serum of
human patients with calcinosis, Raynaud’s phenomenon,
esophageal dysmotility, sclerodactyly, and telangiectasia
(CREST) syndrome and Alexa647-labeled anti-human secondary antibodies for IF. Other anti-centromere antibodies
and secondary antibodies labeled with other dyes that are
optically distinct from MTSb-targeting MBs can potentially
be used.
13. We used DV2-cube (ET525/50 m, 585dcxr, ET655lp, Photometrics) for simultaneous dual-color imaging of telomeres and
centromeres.
14. Other software can potentially be used.
15. HEK293 cells attach and spread more rapidly on fibronectincoated glass surfaces.
16. FuGENE
® 6 was used for transfection in our experiments.
Other transfection regents can potentially be used.
17. Gentle pipetting minimizes cell damage.
18. When low-retention microcentrifuge tubes are used, cell pellets may not form.
19. Microporation parameters are cell line dependent.
Visualizing Genomic Loci by CRISPR/MB
369