Heterochromatic 3D genome organization is directed by HP1a- and H3K9-dependent and independent mechanisms
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These directories contain source images used for all microscopy shown in the main and supplemental figure panels in "Heterochromatic 3D genome organization is directed by HP1a- and H3K9-dependent and independent mechanisms."
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These data are intended to accompany "Heterochromatic 3D genome organization is directed by HP1a- and H3K9-dependent and independent mechanisms." A detailed description of how these data were generated and motivations for doing so are provided there. A brief description of how these experiments were performed is below. Please see the publication for more details about the genotypes used. Immunofluorescence (Figure 2, Figure 3) was done using whole wandering L3 larvae. Cuticles were inverted, fixed in 4% parafomaldehyde, washed with 1xPBS plus 0.1% Triton X-100, incubated with appropriate antibodies (1:1,000 anti-H3K9me3 [Abcam ab8898], 1:1,000 anti-H3K9me2 [Abcam ab1220] and 1:1,000anti-HP1a [Developmental Studies Hybridoma Bank, C1A9]) overnight at 4°C, followed by secondary antibody incubation (1:1,000 Alexa Fluor 647 [Invitrogen MG121], 488 [anti-mouse Invitrogen A-21131 or anti-rabbit Sigma 18772-1ML-F]) for 2-hours at room temperature and DAPI before dissecting and mounting wing discs. Images shown are maximum projections of 3-5 (0.3µm) slices taken at a constant gain and offset using a Leica SP8 confocal microscope with a 64x objective and 2.5x – 3x digital zoom. Western blots (Supplemental Figure 2) were completed using dissected wing disc of 3LW female larvae. Tissues were lysed in 1x Laemmli sample buffer, spun down, supernatant collected and run on a BioRad 4-20% Mini-PROTEAN® TGX™ Precast Protein Gels (456-8094), then transferred to a 0.2um nitrocellulose membrane (BioRad 1620097) and probed with Abcam rabbit anti-B-tubulin (ab60460) and rabbit anti-HP1a kindly provided by Sarah Elgin (M0552). For DNA-FISH (Figure 7), custom designed myTags probes were designed, produced and labeled by Arbor Biosciences. Probe set A (chr2R: 5,060,000-5,190,000) was labeled using ATTO 647 and targets a 130kb pericentromeric region with an average probe density of 24.81 probes/kb; probe set B (chr2R: 8,450,000-8,500,000) was labeled using Alexa 488 and targets a 50kb pericentromeric region with an average probe density of 25.86 probes/kb; and probe set C (chr2R:11,950,000-12,000,000) was labeled using ATTO 550 and targets a 50kb pericentromeric region with an average probe density of 27.67 probes/kb. Wandering L3 larval cuticles were inverted, fixed in 1xPBS with 4% paraformaldehyde for 25 mins, washed in 1xPBS plus 0.1% Triton X-100, treated with RNase A and permeabilized with 1xPBS plus 0.3% Triton X-100. Tissues were denatured at 82°C for 10 mins, probes hybridized at 37°C overnight before dissecting and mounting wing discs.