Menon_et_al_Molecular_Cell_2024
Description
Images used in Menon at al., Proximal termination generates a transcriptional state that determines the rate of establishment of Polycomb silencing, Molecular Cell, 2024 FLC-Venus time-course confocal imaging in Arabidopsis meristem in different genetic backgrounds. Raw images in .tif format are provided for all panels in Figures 5D and Supplemental Data S4, along with additional replicates in some cases. Each subfolder is named after the corresponding genotype.
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Steps to reproduce
Imaging Seeds were surface-sterilized with 5% (v/v) sodium hypochlorite for 5 min, rinsed with sterile water 4x for 1 min and stratified at 4°C for 2 days in water and in the dark. Seeds were plated on MES buffered Murashige and Skoog (GM-MS) media, pH 5.8 and grown on vertically oriented petri plates for 7 days in long-day conditions (16 h light/8 h dark, 22/20°C). Time course imaging of FLC-Venus protein levels in the epidermis of Ler, fca allelic mutants, the clf-2 mutant as well as the fca-3 clf-2 mutant, was performed using a Leica confocal Stellaris 8 microscope with a 20x multi-immersion objective (0.75 NA). Root tips (2 cm) were cut off and immersed in 1.5 µg/mL Propidium Iodide (PI, Sigma–Aldrich, P4864) in 1x PBS before being imaged immediately. The OPSL 514 laser was used at 5% power with 514 nm excitation for FLC-Venus and PI. In photon counting mode, Venus was detected between 518-550 nm with the HyD SMD2 detector, PI was detected between ~600-650 nm. Images were acquired with a laser speed of 400 Hz, line accumulation of 6 (pixel dwell time of 2.43 µs), a Z-step size of 0.95 µm and a pinhole size of 1 AU. The same settings were used at all imaging time points to allow direct comparison between genotypes and time points. The representative images were projected using a single middle slice from the PI channel to show the cell outline, and 10 slices of FLC-Venus channel were maximum intensity projected. In standard image presentation, the dynamic range of the FLC-Venus channel was pushed from 0-255 to 2-11, in enhanced image presentation, the dynamic ranged was pushed from 2-7. In Supplementary Figure S5C, the dynamic range of FLC-Venus channel was enhanced from 0-255 to 2-20 for all images. To confirm the generality of our results for fca-3, we also observed similar behaviour of FLC expression in single cells for a different intermediate mutant of FCA, called fca-424 (Fig.S5C), showing these effects are not allele-specific, but related to the regulatory function of FCA at FLC.