Extended Data Fig. 3: Worm sorting and quantification settings based on gating region.

a, COPAS Biosort conditions optimised for the quantification of the heterochromatic reporter fluorescence. The COPAS Biosort (Union Biometrica) machine is an adapted flow cytometry version that can be used in order to quantify and collect worms according to their size and fluorescence criteria. The upper panel reflects the gating region (black diamond) based on the optical density of the object (optical extinction, EXT in the y axis) and the axial length (time of flight, TOF, in the x axis) of the object selecting the L1 worm population, as determined empirically in pilot experiments by verifying the stage through microscopic examination of sorted worms with this gate criteria. The same criteria gating was identical for every quantification of the heterochromatic reporter fluorescence. The lower panel shows the worm distribution of the size-selected worms based on green parameters (green peak height (green PH) and green peak width (green PW). This lower panel in a, represents the fluorescence of the heterochromatic reporter (GW306) in control RNAi condition and in b, in lsm-7 RNAi conditions. c, COPAS Biosort conditions optimised for the sorting of homozygous lsm-8 mutant at the L3 stage. The upper panel reflects the gating region (black diamond) based on the optical density of the objects (optical extinction, EXT in the y axis) and the axial length (Time of Flight, TOF, in the x axis) of the objects selecting the L3 worm population. The lower panel shows the worm distribution based on green parameters (green peak height (green PH) and green peak width (green PW)), the second gating region (black window) shown in that panel selects here the non-green worms, homozygous for lsm-8. The gating strategies were determined empirically in pilot experiments by verifying the size, shape gonad and vulva developmental stage by microscopic examination. Morphological validations during the sorting process were also performed. Sorting of the homozygous animals was done by selecting non- GFP pharynx animals, and the gating was also determined stringently by examining the two populations and by verifying the different criteria with fluorescent microscopy.