Fig. 2

Characterization of 27nt-RNAs and their association with macronuclear sequences. a Time course of small RNA fragment size distribution (10–50nt) and associated stages of macronuclear development showing the dominance of 27nt—RNAs (light blue columns). Red columns mark 21–22nt-RNAs. The y-axis shows the normalized read quantity (× 10⁵). b The illustration shows the coverage of 27nt-RNAs (light blue bars), 21–22nt-RNAs (red bars) and mRNA reads (green curves) on four exemplary nanochromosomes with complete telomere-to-telomere sequence (red contigs). Notably, the mapped area frequently includes introns (1.95% of 27nt-RNA/1.43% of 21–22nt-RNA). The position of predicted full-length transcripts (yellow arrows) and coding sequences (CDS; green arrows, occasionally interrupted by introns [red arrows]) is illustrated. The bars below (purple) show furthermore the coverage of 27nt-RNAs, which were purified from pulled-down RNA/DNA hybrids using the mouse anti-RNA/DNA hybrid [S9.6] monoclonal antibody (mAb). c Average sncRNA (27nt-RNAs and 21–22nt-RNAs) read counts matching per contig. d Combined correlation analyses between mRNA reads (transcript), 27nt-RNAs and the relative nanochromosome copy numbers. e PAR-CLIP revealed a prominent RNA band well in agreement with the expected size of a PIWI1/27nt-RNA complex (sncRNA1)), whereas a smaller, at best very faint band could correspond to the expected size of a PIWI1/21–22nt-RNA complex (sncRNA2). f The read coverage is shown on the left for 4 exemplary nanochromosomes for 27nt-RNAs enriched in immunocomplexes using the mouse anti-RNA/DNA hybrid [S9.6] mAb (Kerafast #ENH001) (purple signals) or, respectively, rabbit anti-PIWIL1 polyclonal antibody (pAb) (Abcam #ab12337) (light blue signals). Symbolism and colour coding of the nanochromosome annotation equals (b). The chart (right) shows the results of correlation analyses between the reads obtained from 27nt-RNA-seq (x-axis: RNA/DNA-IP; y-axis: PIWI1-IP)