Fig. 2
Purification of new developing MACs from Paramecium tetraurelia by flow cytometry and validation by flow cell imaging and high throughout DNA sequencing. a. DAPI staining of a cell upon PGM RNAi at a late developmental stage of the sexual process of autogamy (self-fertilization) is shown on the picture: the two large new developing MACs (dotted circle) and the small fragments of the maternal MAC are detected. The scale bar is 10 microns. b. Following gentle lysis and cell fractionation, the nuclei preparation is submitted to flow cytometry after staining with DAPI. c. Multi-gating flow cytometry strategy used for sorting. Sorting is based on size, granularity and DAPI staining signal of the new developing MACs. An empiric iterative procedure coupled with flow imaging allowed discrimination between developing MACs and fragments, identification of the population of interest, and optimization of the sorting strategy. d - e. The Amnis ImageStreamX imaging flow cytometer is used for quality control. Distribution of DAPI intensity is shown for each event in the sample before (d) and after sorting (e), respectively. Representative images are displayed in BF (bright field) and DAPI. Objective ×60. f. Validation of the sorting strategy by high-throughput DNA sequencing. Histograms of IES retention scores are shown for control (no RNAi), PGM RNAi (no sorting) and PGM RNAi after sorting (flow cytometry)