Analyses of sRNA-seq libraries.(A) Hierarchical clustering analysis showing the degree of similarity among the sRNA-seq libraries. Sample-to-sample distances were calculated based on log-transformed normalized read counts. The biological replicates of each sample type were highly reproducible. T: total extract; M: microsome; TP: total polysome; MBP: membrane-bound polysome; T27: sRNA-seq from total extracts in ago1-27; M27: sRNA-seq from microsomes in ago1-27. The other samples were from wild type. The numbers after the underscore represent biological replicates. (B) Size distribution of sRNAs in the indicated samples. T: total extract; M: microsome; ER-IP: ER immunoprecipitation. Note that normalization was performed against total r/t/snRNA-depleted reads. (C) The composition of sRNAs in each of three size classes. Each column represents a biological replicate. Note that normalization was performed against total r/t/snoRNA-depleted reads. (D) Western blotting using anti-GFP antibodies to show that YFP-SEC12 was in the microsomal fraction. (E) Topology mapping showing that the YFP tag of the ER transmembrane protein YFP-SEC12 was on the cytosolic side. (F) Western blotting showing that ER immunoprecipitation (using anti-GFP antibodies with the YFP-SEC12 transgenic line) contained two ER markers (YFP-SEC12 and HSC70) but not markers for Golgi (ARF1) or endosomes (SYP22). PEPC is a soluble protein. Col, wild type (without the YFP-SEC12 transgene). (G) A scatter plot showing the depletion of Pol IV-dependent siRNAs (P4siRNAs) from the microsomal fraction. Each dot represents a 100 bp window that generates P4siRNAs. Note that normalization was performed using rRNA fragments as internal controls. (H–K) Wild type and nrpd1-1 sRNA-seq samples that differ greatly in sRNA compositions were used as a proof-of-concept for the rRNA fragment-based normalization method. (H–I) Normalization using total r/tRNA-depleted, genome-matched reads led to an exaggeration of the 21-nt peak (H) and an apparent increase in the abundance of miRNAs (I) in nrpd1-1, which lacks most 24-nt siRNAs. (J–K) Normalization using rRNA fragments resulted in similar abundance of 21-nt siRNAs (J) and miRNAs (K) between wild type and nrpd1-1.DOI:http://dx.doi.org/10.7554/eLife.22750.003

Release of CrPAM in ciliary ectosomes is developmentally regulated.A. HAP2 minus and CC125 plus gametes, mating cells (0- or 1-hour), and ectosome-rich pellets prepared from the 1-hour media were analyzed. Equal amounts of protein (30 ?g) were fractionated and subjected to immunoblot analysis for ARF1, FMG1, and CrPAM. Quantification of FMG1 and CrPAM protein levels is shown in the lower panels; results are the average of two independent experiments—error bars indicate the range. B. Cell lysates and ectosomes prepared from wild-type CC124?/CC125+ gametes were analyzed as described for panel A. Results are the average of six independent experiments; error bars indicate ± SEM. Asterisks indicate a statistically significant difference between two groups (*P < 0.01). C. and D. Immunoblot analysis of cells and ectosomes harvested from vegetative CC124? (C) and CC125+ (D) cells; quantification of FMG1 and CrPAM levels is shown below. Results are the average of three experiments; error bars indicate ± SEM. CrPAM and FMG1 levels in vegetative ectosomes differed significantly from levels in cells (**P = 0.0065, ***P < 0.0001). E. CrPHM and CrPAL activities were assayed in cells and ectosomes released by vegetative CC124? and CC125+ cells, mating HAP2?/CC125+ cells, and mating CC124?/CC125+ cells. Both activities were significantly higher in ectosomes released by mating gametes (*P < 0.01, **P < 0.001, ***P < 0.0001; one-way ANOVAs). F. Immunogold-electron microscopy negative stain image showing localization of CrPAM on mating ectosomes with antibody against CrPAM luminal domain. Negative control, ectosomes incubated with gold-tagged secondary antibody alone. The underlying numeric data for this figure can be found in S1 Data. ARF1, ADP ribosylation factor 1; FMG1, flagellar membrane glycoprotein 1.