Fig. 3
Purification of germline MICs from vegetative Paramecium tetraurelia by flow cytometry and validation by flow cell imaging and high throughout DNA sequencing. a. In CENH3a-GFP transgenic Paramecium vegetative cells (upper panels), but not in control cells (lower panels), the MICs are GFP positive. Scale bar is 10 microns. Higher magnification: Scale bar is 3 microns. b. Fractionation scheme used to isolate the MIC-enriched fraction. c - d. Multi-gating strategy used for sorting the MICs. Sorting is based on size, granularity and DAPI staining and GFP signals in c) CENH3a-GFP transgenic cells and d) control cells. An empiric iterative procedure coupled with flow imaging allowed discrimination between MICs and DAPI containing contaminants, identification of the population of interest, and optimization of the sorting strategy. e - f. The Amnis ImageStreamXimaging flow cytometer is used for quality control: sample before (e) and after sorting (f). g. Validation of the sorting strategy by high-throughput DNA sequencing. Histograms of IES retention scores are shown for control (no RNAi), MIC (no sorting) and MIC after sorting (flow cytometry)