Fig. 7. Detection of circRNA junction peptides using LC-MS.

a Workflow for assembling junction peptide sequence database. RNA-seq data were processed by CIRI2 to map the circRNA junction site to the mouse genome. The circRNA 3′ (pink) and 5′ back splicing site (dark) are joined with 60 bp adjacent sequences (dark green). The predicted, trypsin-digested circRNA junction peptide sequences were collected in the circRNA junction peptide sequence database, which will be used for analyzing LC-MS data. b Number of junction peptides identified by LC–MS in pachytene spermatocytes, round, and elongating spermatids, as well as spermatozoa. Bars represent the total number of junction peptides identified from LC-MS analyses in three replicates (numbers of junction peptides from three biological replicates were combined). c Close correlations between counts of junction peptides identified via LC–MS and those of circRNA junction sequences identified from RNA-seq in pachytene spermatocytes, round, and elongating spermatids, as well as spermatozoa (R² = 0.94, P = 0.05, counts of three biological replicates were combined for analyses). d Examples of two circRNA junction peptides identified using LC–MS. The junction peptide sequences are fully covered. The relative intensity of each peptide identified is indicated in each vertical line. The y-axis shows the relative ion intensity and x-axis shows the mass to charge ratio (m/z). Peptide fragment ion in the spectrum is labeled in red. Peptide fragment ion missing from the spectrum is labeled in black.