Supplementary Figure 1. Alternative splicing and cirexon detection in CIRI-AS. (A) Split-alignment of BSJ read pairs in detecting exon skipping,

Transcript

1 Supplementary Figure 1. Alternative splicing and cirexon detection in CIRI-AS. (A) Split-alignment of BSJ read pairs in detecting exon skipping, alternative 5 /3 splicing site and intron retention events. (B) Sequencing depth variation and non-bsj read pairs are used to detect FSJs and cirexons missed by BSJ-read pairs. (C) Impact of library insert sizes and variances as well as sequencing depth on FSJ detection by BSJ read pairs.

2 Sensitivity/FDR Sensitivity/FDR A 1.00 B Insert length distribution 0.50 Read length 75 bp bp 125 bp 150 bp C Sequencing depth Supplementary Figure 2. Performances of CIRI-AS for cirexon detection on simulated data sets. (A) Sensitivity and FDR for cirexon detection with varying sequencing depths in simulated data sets (PE100 bp) for four distributions of insert lengths. (B) Sensitivity and FDR for cirexon detection with varying sequencing depths in simulated data sets (read length distribution parameter: μ 1=280 bp, σ 1=80 bp, μ 2=370 bp, σ 2=100 bp, p=0.9) for four sequencing read lengths. D Sequencing depth

3 A B 421 nt D 176 nt E 199 nt G C 487 nt F 355 nt 369 nt 326 nt J 361 nt I 447 nt Supplementary Figure 3. Examples of validated cirexons detected by CIRI-AS using long-read data. Detected cirexons by CIRI-AS were shown on left and the corresponding components identified in long-read data were shown on right.

4 A 362 nt B 367 nt C 325 nt D 267 nt E 337 nt F 320 nt G 174 nt H 363 nt Supplementary Figure 4. Examples of cirexons identified using long-read data but not detected by CIRI-AS using bp paired reads. Detected cirexons by CIRI-AS were shown on left and the corresponding components identified in long-read data were shown on right. Inner colored solid-line curves indicate the cirexons detected by CIRI-AS, whereas the dashed-line curves represent the cirexons missed by CIRI-AS.

5 A B C 246 nt 246 nt 207 nt 311 nt 413 nt 397 nt D 179 nt E 164 nt 228 nt F 258 nt 343 nt G 208 nt 354 nt 284 nt Supplementary Figure 5. Application of CIRI-AS on bp paired reads can detect more isoforms than low-coverage long-read based identification. Detected cirexons by CIRI-AS were shown on left and the corresponding components identified in long-read data were shown on right. The internal compositions of alternatively splice transcripts not identified in long-read data were shown in the left bottom.

6 Relative abundance most abundant second abundant other isoforms Absolute abundance all isoforms #BSJ reads % of isoforms Supplementary Figure 6. The absolute abundances of top 50 most abundant alternatively spliced circrnas as well as relative abundances of their AS transcripts detected in RNase R+ sample of HeLa cells.

7 A 81,737,400 CMIP (16q23.2) 81,738,200 81,739,000 81,738,800 81,741,000 binding motif: GCUGGCC hsa-mir p binding sites circcmip_1 circcmip_2 sequencing depth 147bp 1413bp 106bp 1409bp 71bp B TPCN1(12q24.13) 113,705, ,706, ,706, ,706, ,707, ,707,600 binding motif: AGAGUGG hsa-mir p binding sites circtpcn1_1 circtpcn1_2 sequencing depth 114bp 785bp 131bp 884bp 89bp Supplementary Figure 7. Examples of the density of mirna binding sites within circrnas altered by AS events.

8 Supplementary Figure 8. Circular transcripts with exon skipping validated by RT-PCR and Sanger sequencing in HeLa cells. For the FSJs in transcripts with exon skipping, only related BSJ read pairs were shown. Reads in poly(a) selected sample of HeLa cells that indicate the corresponding splice junctions were shown in the right column.

9 Supplementary Figure 8. (continued)

10 Supplementary Figure 9. Circular transcripts with alternative 3 splicing site validated by RT-PCR and Sanger sequencing in HeLa cells. For the FSJs in transcripts with alternative 3 splicing site, only related BSJ read pairs were shown. Reads in poly(a) selected sample of HeLa cells that indicate the corresponding splice junctions were shown in the right column.

11 Supplementary Figure 10. Circular transcripts with alternative 5 splicing site validated by RT-PCR and Sanger sequencing in HeLa cells. For the FSJs in transcripts with alternative 5 splicing site, only related BSJ read pairs were shown. Reads in poly(a) selected sample of HeLa cells that indicate the corresponding splice junctions were shown in the right column.

12 Supplementary Figure 11. Circular transcripts with intron retention validated by RT-PCR and Sanger sequencing in HeLa cells. For the FSJs and retained introns in the transcripts, only related BSJ read pairs were shown. Reads in poly(a) selected sample of HeLa cells that indicate the corresponding splice junctions were shown in the right column.

13 Supplementary Figure 12. Resistence of circular AS isoforms to RNase R quantified by qrt-pcr. (A-D) Nine circular AS isoforms of four circrna loci (n = 3; mean ± SE). (E) Linear control of GAPDH and Actin (n = 3; mean ± SE). Red bars represent total RNA samples, whereas blue bars represent RNase R-treated RNA samples.

14 A B C Supplementary Figure 13. Three examples of circular AS isoforms detected by both CIRI-AS (upper panel) and long-read sequencing (lower panel). In the lower panel, blue lines indicate the long reads that can cover the full length of circrnas. Curved lines indicate the splicing junctions. Black triangles indicate BSJs located in the long reads.

15 Supplementary Figure 14. Performances of CIRI-AS for relative abundance estimation on simulated data sets. (A) Estimated Ψ values without correction or after correction in CIRI-AS for exon skipping within simulated circrnas with three simulated Ψ values. (B) Estimated Ψ values without correction or after correction in CIRI-AS for exon skipping within simulated circrnas with four sequencing depths.

16 Supplementary Figure 15. Relative abundance of nine AS isoforms in four circrna loci estimated by qrt-pcr and CIRI-AS. (A) Relative abundance of nine AS isoforms in four circrna loci estimated by qrt-pcr (n = 4; mean ± SE); (B) Relative abundance of nine AS isoforms in four circrna loci estimated by CIRI-AS; (C) Comparison of relative abundances estimated by CIRI-AS and qrt-pcr (n = 4; mean ± SE).

17 10% 1% ICF 8% overlapped_with_known_exons 16% 54% protein_coding processed_transcript 11% nonsense_mediated_decay retained_intron Supplementary Figure 16. Compositions of cirexons with higher relative abundance in circrnas compared to mrnas in HeLa and HEK293. Cirexon classification is based on the Gencode annotation.

18 Supplementary Figure 17. Exon skipping events detected in mouse immune dendritic cells with 10 min labeling pulse of 4-thiouridine. (A) Comparison of Ψ value for exon skipping events between circrna and mrna. (B) Information about skipped cirexons including read counts, parent gene names of circrnas, position of cirexons as well as P values after correction for multiple testing (corrected P values <0.10 are shown in bold). Skipped cirexons classified as ICFs are shown in red, and other skipped cirexons in blue.

19 CNOT4 CNOT4.1 RBM5 FMR1 FMR1.1 RBM38 ANKHD1 BRUNOL6 TARDBP HNRNPM BRUNOL4 BRUNOL5 HNRNPU ZNF638 RBM24 PCBP3 RBM28 SNRNP70 G3BP2 RBFOX1 KHDRBS2 PABPC4 KHDRBS3 RBMS3 A1CF ZCRB1 KHDRBS1 PUM2 RBM41 PABPC5 DAZAP1 QKI RBMS1 CPEB4 HuR HNRNPC HNRNPCL1 TIA1 ZC3H14 CPEB2 RALY U2AF2 PTBP1 TUT1 PABPN1 HNRNPL HNRNPL.1 HNRPLL YBX1 IGF2BP2 IGF2BP3 FXR1 RBM45 RBM6 ENOX1 SNRPA SRSF10 MATR3 PABPC3 PABPC1 SART3 RBM3 RBM42 MSI1 SFPQ RBM46 YBX2 SRSF1 SRSF7 HNRNPK RBM8A FUS PPRC1 HNRNPF HNRNPF.1 SAMD4A SRSF2 MBNL1 HNRNPH2 RBM4 ZC3H10 FXR2 HNRNPH1 LIN28A ESRP2 HNRNPA1L2 SRSF9 SRSF9.1 HNRNPA2B1.2 HNRNPA2B1 HNRNPA2B1.1 HNRNPA1.2 HNRNPA1 HNRNPA1.1 PCBP1 PCBP skipped cirexon skipped cirexon constitutive constitutive skipped skipped constitutive constitutive randomly (#BSJs >20) cirexon cirexon mrna exon mrna exon mrna exon mrna exon selected exon (#BSJs > 20) (FPKM > 5) (FPKM > 5) Supplementary Figure 18. Comparison of binding site density of RNA binding proteins on skipped cirexons, constitutive cirexons, skipped mrna exons in HeLa cells and randomly selected annotated exons. The binding numbers were normalized by corresponding exon length to calculate average binding density, which was shown in the heatmap after Z-score normalization.

20 skipped cirexon skipped cirexon (#BSJ reads>20) constitutive cirexon constitutive cirexon (#BSJ reads>20) skipped mrna exon skipped mrna exon (FPKM>5) constitutive mrna exon constitutive mrna exon (FPKM>5) randomly selected exon Supplementary Figure 19. Total number of exons in each group used for splicing factor binding site prediction. Colors of each group correspond to those in Fig 3A.

21 % of AS exons in nuclear circrnas 1.25% Type 1.00% 0.75% 0.50% 0.25% A3SS A5SS ES RI as.factor(cell_line) GM12878 HepG2 0.00% 0.00% 0.25% 0.50% 0.75% 1.00% 1.25% % of AS exons in cytosolic circrnas K562 Supplementary Figure 20. A comparison between nucleus and cytoplasm for percentage of four types of alternatively spliced cirexons in HepG2, K562 and GM12878 cell lines. All of the three cell lines have comparable sequencing data size in nucleus and cytoplasm.

22 % of AS exons in nuclear circrnas % of AS exons in nuclear mrnas A 0.4% B as.factor(cell_line) 1.5% as.factor(cell_line) GM12878 GM % 0.2% H1 hesc 1.0% HeLa S3 HepG2 H1 hesc HeLa S3 HepG2 0.1% HUVEC 0.5% K562 HUVEC K % 0.0% 0.1% 0.2% 0.3% 0.4% % of AS exons in cytosolic circrnas NHEK 0.0% 0.0% 0.5% 1.0% 1.5% % of AS exons in cytosolic mrnas NHEK Supplementary Figure 21. A comparison between nucleus and cytoplasm for percentage of retained intron in seven cell lines. (A) percentage of retained intron in circrnas; (B) percentage of retained intron in mrnas.

23 A PolyA & Nucleus PolyA & Cytoplasm PolyA+ & Nucleus PolyA+ & Cytoplasm B PolyA & Nucleus PolyA & Cytoplasm PolyA+ & Nucleus PolyA+ & Cytoplasm Supplementary Figure 22. Examples of intron retention within circrna observed in poly(a)- sequencing data set of nucleus but not in cytoplasm. Sequencing depths of mrna observed in corresponding poly(a) selected sequencing data sets of both nucleus and cytoplasm are shown. (A) Retained intron 2 in circap3s2; (B) Retained intron 8 in circclk1.

24 PC 1 [ 27 %] PC 2 [ 5 %] PC 3 [ 4 %] PC 4 [ 4 %] Supplementary Figure 23. Principal component analysis based on the Ψ values of the AS cirexons in fruit fly. Each dot represents a sample and its color corresponds to the sample type on the top of Figure 4A. The first four principal components were displayed.

25 Supplementary Figure 24. Relative abundance of alternatively spliced cirexons in 18 fruit fly eclosion samples.

26 Supplementary Figure 25. Relative abundance of skipped cirexons detected in circrnas compared with corresponding mrnas in three fruit fly samples (eclosion + 1 day, heads).

27 #BSJ of circrna log10(polya FPKM) Supplementary Figure 26. Number of BSJ reads for circrnas and FPKM of the corresponding linear RNAs in HEK293 and HeLa cell lines. Red dot: HeLa cell line; Black dot: HEK293 cell line.

28 Supplementary Table 1. Data sets of human samples used in this study for cirexon/exon detection Cell type Treatment Read Insert size (bp) Data (Gb) # BSJs # cirexons # ICFs Data source (SRA accession #) HeLa RiboMinus PE ± ,606 11, This study (SRR , SRR & SRR ) HeLa RiboMinus & PE ± ,631 33,241 2,755 This study (SRR , RNase R+ SRR & SRR ) HeLa PolyA+ PE ± n/a n/a n/a This study (SRR ) HEK293 RiboMinus PE ± ,916 8, This study (SRR ) HEK293 RiboMinus & PE ± ,465 33,650 2,453 This study RNase R+ (SRR ) HEK293 PolyA+ PE ± n/a n/a n/a This study (SRR ) Hs68 RiboMinus PE ± ,532 3, Jeck et al., 2013 (SRR444655) Hs68 RiboMinus PE ± ,387 7,475 1,089 Jeck et al., 2013 Hs68 Hs68 RiboMinus & RNase R+ RiboMinus & RNase R+ (SRR444975) PE ± ,685 54,785 6,735 Jeck et al., 2013 (SRR445016) PE ± ,537 53,974 5,702 Jeck et al., 2013 (SRR444974)

29 Supplementary Table 2. Exon skipping in top 50 most abundant circrnas of HeLa cells. circrna cirexon Psi value #BSJ reads chr9: : chr9: : chr2: : chr2: : chr2: : chr2: : chr2: : chr21: : chr21: : chr3: : chr3: : chr3: : chr3: : chr7: : chr15: : chr15: : chr15: : chr15: : chr15: : chr21: : chr21: : chr21: : chr21: : chr3: : chr5: : chr12: : chr12: : chr8: : chr8: : chr8: : chr8: : chr8: : chr8: : chr2: : chr2: : chr2: : chr2: : chr2: : chr8: : chr8: : chr8: : chr8: : chr4: : chr3: : chr9: : chr12: : chr12: : chr10: : chr21: : chr3: : chr3: :

30 chr21: : chr1: : chr5: : chr22: : chr20: : chr9: : chr17: : chr17: : chr5: : chrx: : chrx: : chrx: : chr5: : chr7: : chr7: : chr19: : chr11: : chr11: : chr20: : chr13: : chr13: : chr13: : chr10: : chr9: : chr6: : chr2: : chr2: : chr2: : chr2: : chr9: : chr21: : chr21: : chr12: : chr14: : chr9: : chr11: : chr21: : chrx: : chr8: : chr8: :

31 Supplementary Table 3. Alternative 5 /3 splicing site events in top 50 most abundant circrnas of HeLa cells. circrna cirexon Psi value #BSJ reads chr9: : chr9: : chr9: : chr9: : chr1: : chr1: : chr10: : chr10: : chr10: : chr10: : chr2: : chr2: : chr2: : chr2: : chr2: : chr5: : chr5: : chr5: : chr5: : chr3: : chr3: : chr3: : chr3: : chr15: : chr15: : chr15: : chr15: : chr15: : chr5: : chr5: : chr5: : chr5: : chr5: : chr21: : chr21: : chr21: : chr21: : chr21: : chr21: : chr3: : chr3: : chr5: : chr5: : chr12: : chr12: : chr12: : chr12: : chr8: : chr8: : chr8: : chr8: : chr2: : chr2: : chr2: : chr2: : chr8: :

32 chr8: : chr8: : chr8: : chr8: : chr12: : chr12: : chr18: : chr18: : chr10: : chr10: : chr9: : chr9: : chr3: : chr3: : chr9: : chr9: : chr12: : chr12: : chr2: : chr2: : chr22: : chr22: : chr22: : chr22: : chr22: : chr22: : chr9: : chr9: : chr9: : chr2: : chr2: : chr2: : chr2: : chr2: : chr2: : chr7: : chr7: : chr15: : chr15: : chr19: : chr19: : chr19: : chr7: : chr7: : chr11: : chr13: : chr13: : chr6: : chr6: : chr1: : chr1: : chr14: : chr14: : chr2: : chr2: : chr14: : chr14: : chr9: : chr9: : chr21: : chr8: :

33 chr8: : chr16: : chr16: : chr8: : chr8: : chr1: : chr1: : chr1: : chr1: : chr10: : chr10: : chr10: : chr10: : chr17: : chr17: : chr6: : chr6: : chr1: : chr1: : chr4: : chr4: :

34 Supplementary Table 4. Intron retention in circrnas of HeLa cells. circrna cirexon Psi value #BSJ reads chr9: : chr5: : chr7: : chr12: : chr8: : chr15: : chr19: : chr10: : chr10: : chr11: : chr1: : chr19: : chr2: : chr1: : chr19: : chr2: :

35 Supplementary Table 5. Increased mirna binding sites within skipped cirexons in Hs68 cell line. mirna cirexon(start:end) within circrna(chr:start end) hsa-mir p : chr1: hsa-mir p : chr10: hsa-mir p : chr11: hsa-mir p : chr13: hsa-mir p : chr13: hsa-mir : chr13: hsa-mir p : chr13: hsa-mir p : chr13: hsa-mir : chr17: hsa-mir-15a-5p : chr17: hsa-mir-15b-5p : chr17: hsa-mir-497-5p : chr17: hsa-mir p : chr2: hsa-mir p : chr2: hsa-mir : chr5: hsa-mir-330-5p : chr9: hsa-mir : chr9: Length_ mirna Length_ cirexon Postions_binding_ sites

36 Supplementary Table 6. circrna producing loci with AS events validated in RT-PCR of this study and corresponding outward-facing primers. chr start position end position P1 outward-facing primer P2 isoform length (bp) PCR & Sanger chr GTGCTACTTTTCCTAATAGAAC TCTCTTATATGCATCAAGTCTA 844 y chr TTGGGCAATGGTGAATTTGAA AGGTGTGTTAAAGGCAGGAA 648 y chr GTTCGCAGTCTGAGTCATGGAA ACCCTGTGACTCCTTGCTTT 382 y chr GTTCGCAGTCTGAGTCATGGAA ACCCTGTGACTCCTTGCTTT 304 y chr GTTCGCAGTCTGAGTCATGGAA ACCCTGTGACTCCTTGCTTT 242 y chr GAGCTTCATGGTGGAGAAGGG CGCAGCCAGAAAGCCTCC 319 n chr GCAGTATAGAGACGTCGGACT TGTCCGGGTGGAAATCCGTGA 242 y chr CAGTGACATCTTAGCTGCTA CGGTATCTTGCCAATCACTGTA 165 y chr CAGGGGAAACTTGAACATTCG CACCAATACCTAAGACAGAGCAG 257 y chr AGTGACCTGGACAAGAGT TGCTGCCGCTCGTAATATCT 344 y chr GACGTGGAGAAGTTCATGCCAA TCGTTCCGGTCTTGGAGTGTCA 326 y chr GTGACCTGGACAAGTACATCATT TGCTGCCGCTCGTAATATCT 284 y chr CGTTCAACTCCAGCAATGATGAAT ACTGGAAATTGTGCTGTCTACAT 419 y chr CGTTCAACTCCAGCAATGATGAAT ACTGGAAATTGTGCTGTCTACAT 316 y chr CCAAGTTCCAGGATTAATTCTG GTCACATAAAGGAGGGTTAGAA 409 y chr CCAAGTTCCAGGATTAATTCTG GTCACATAAAGGAGGGTTAGAA 332 y chr CCAAGTTCCAGGATTAATTCTG GTCACATAAAGGAGGGTTAGAA 272 n chr AGTGGACGTACGGGGATTCAT ACACATTCAGCAGCAAGGAG 251 y chr TGAACAATGACGGCAATCTTGT GAGGGGACTTGATGTTAACAAA 395 n chr AGTGGACGTACGGGGATTCAT ACACATTCAGCAGCAAGGAG 274 y chr TTGCAGACAGCGGATGAAACTTA ACTGCAGGTGGTATTAAATTTGTG 328 y chr AAGCTAAAAATCAGGCAACACC GCATCTGATATCTGGAAACTGAA 398 y chr AAGCTAAAAATCAGGCAACACC GCATCTGATATCTGGAAACTGAA 496 y chr TTGCAGACAGCGGATGAAACTTA ACATCATGAACTGCAGGAGTG 426 y chr CTGCTGCAAAATGGTATCATTC ATTGGTATCACAGTGCACCT 338 y chr CTGCTGCAAAATGGTATCATTC ATTGGTATCACAGTGCACCT 408 y chr CTGCTGCAAAATGGTATCATTC ATTGGTATCACAGTGCACCT 436 y chr TCTGTATCCTTTTCCTATGGC TGGTGGAAATTTGATCCTGA 438 y chr TCTGTATCCTTTTCCTATGGC TGGTGGAAATTTGATCCTGA 388 y chr GTGCTGGAATTCTGCATCGTAGGT TGAGATTGTCCTGAGTGCTGGCT 280 n chr GTGCTGGAATTCTGCATCGTAGGT TGAGATTGTCCTGAGTGCTGGCT 251 n chr CAGGTGCATAGGTCTTGAAGCG TTCCGGGAGCTCTTTGGGAT 249 y chr CAGGTGCATAGGTCTTGAAGCG TTCCGGGAGCTCTTTGGGAT 403 y chr GCTCTCAATGAAGTTATCAGTCGGC TCTGGAACTTTGACGCCCTCTT 300 y chr GCTCTCAATGAAGTTATCAGTCGGC TCTGGAACTTTGACGCCCTCTT 338 y chr TAACGGGCCATTTTCTTGA GCTACTTCCATATATGTGCTT 456 y chr GACCAGATCCATAAAACAGA GACTGAATTCAAGGGAAGAA 407 y chr TGTCATCCGGAGTCTTCAGATT AGAGCTACTTTTCAACAGCCTT 203 y chr ACATTCCAGAAAACGCAAATTC GTCTGGCTTCTTGCACTCAG 284 y chr GCAACTGTGGATGAAGAGGGCT TTCGGCTGTGTTCTCTGGGTTA 231 y chr GGTGAAGGCATTGATTGTCCTGA AACGAGGGCAGAGGCTCATGAT 269 y chr CGAGGCTTCTGGTGTTTGCTTT TGCTGGTGGTGTACTCTGCTAAT 380 y chr CGAGGCTTCTGGTGTTTGCTTT TGCTGGTGGTGTACTCTGCTAAT 256 y

37 chr TGGTGAGGCATACTTCGATCTT TTGCTGCAGATGAAGTGTTACA 683 y chr TGGTGAGGCATACTTCGATCTT TTGCTGCAGATGAAGTGTTACA 566 y chr TGGTGAGGCATACTTCGATCTT TTGCTGCAGATGAAGTGTTACA 487 y chr ACGATGAGCAGCTAAAATGACA CCCATGCAGTTGGCTGTGAAT 460 n chr ACGATGAGCAGCTAAAATGACA CCCATGCAGTTGGCTGTGAAT 358 n chr GCATCTCAGGTCCATGAACTTG AGTCCTCGAGCTTTGACCTTCAT 578 n chr GCATCTCAGGTCCATGAACTTG AGTCCTCGAGCTTTGACCTTCAT 197 n chr CAGGTTTGGTTTTGCTGACCGC GGTGATCGAGGCCTATGGACAT 220 y chr TGGTTTTGCTGACCGCCACTAG CCTGAAGCCCTTTGGAAGCAT 297 y chr CAGGTTTGGTTTTGCTGACCGC GGTGATCGAGGCCTATGGACAT 220 y chr TGGTTTTGCTGACCGCCACTAG CCTGAAGCCCTTTGGAAGCAT 297 y chr GAGTTGGCTTCTGAGGCTTGACT ATTCGCAACACAACAATCAGATGG 377 y chr GAGTTGGCTTCTGAGGCTTGACT ATTCGCAACACAACAATCAGATGG 309 y chr AGCCAGTAGGACACGAAGGT TCAGCTGTGCCCATGACTTTG 226 y chr AGCCAGTAGGACACGAAGGT TCAGCTGTGCCCATGACTTTG 176 y chr TCTCCCTCTGCCACGTCCAAAT AAGGCATGGGGACATTTAATCCT 159 y chr TCTCCCTCTGCCACGTCCAAAT AAGGCATGGGACTATTCAGATTCT 142 y chr ATGAGCGTTCTGGGTCAGG TGGTGTCCCTGTAAGGCGG 587 y chr ACCCATAAAAATGTATCCCACCC TGGTCCCCTTTCAGGATGAGG 639 y

38 Supplementary Table 7. circrnas AS isoforms validated and quantified in qrt-pcr of this study and the corresponding primers used. chr start position end position P1 outward-facing primer chr GCCAGATGATTACTTGACCACCTCCT TGAGGTTGCTGCCTTCGCTGGGGAA 249 chr AGGAGGTGGTCAAGTAATCATCTGGC TTCTTCCCCAGCTCTGTGACCAGT 403 P2 isoform length (bp) chr GCAGCTCTCAGTTTAAACATCT GGAAATGAATTCTAAGGTACTTCGT 256 chr CTCCAAACACAGCAAACTCTCA A GGAAATGAATTCTAAGGTACTTCGT 380 chr AAGGCATGGGACTATTCAGATTCT A CGTCCAAATCCAGTTCCCTCAT 142 chr AAGGCATGGGGACATTTAATCCTG CGTCCAAATCCAGTTCCCTCAT 158 chr AGTAAATATCTAACTCTTCATTTGTGC TTTTCAACAATGCTTCAGAGTCATC 382 chr T TTCAAAAAGGATGACTCTGAAGCATT CAGCACAAATGAAGAGTTAGGCTTA 304 chr CAAGACATAAGCAATAGCTAACTCTTC TTTCAACAATGCTTCAGAGTCATCC 242

39 Supplementary Table 8. Significance of Mann Whitney U tests after FDR-correction for comparison of splicing factor binding density between groups of exons. cires~ cires20 cires~c cires20 mes~ mes5~ cires~r cires20 mes~r mes5~ mes ~mes5 irconst ~circon mconst mconst and ~rand and rand st20 5 FOX1 4.3E E E E E E E E E E-31 Tra2alpha 2.4E E E E E E E E E E-03 SRp55 1.5E E E E E E E E E E-10 Tra2beta 5.9E E E E E E E E E E-36 9G8 4.5E E E E E E E E E E-02 SC35 3.4E E E E E E E E E E-01 QK1 8.8E E E E E E E E E E-29 MBNL 4.4E E E E E E E E E E-09 NOVA1 5.0E E E E E E E E E E-04 hnrnpm 1.4E E E E E E E E E E-59 SF2ASF 3.1E E E E E E E E E E-03 SRp40 6.5E E E E E E E E E E-12 YB1 2.7E E E E E E E E E E-01 hnrnpa1 6.8E E E E E E E E E E-05 hnrnpu 2.0E E E E E E E E E E-73 hnrnpa2b 1.2E E E E E E E E E E-10 1 PTB 4.4E E E E E E E E E E-31 hnrnph1 3.4E E E E E E E E E E-02 hnrnpf 1.1E E E E E E E E E E-10 SRp20 1.2E E E E E E E E E E-02

40 Supplementary Table 9. Intron retention events detected in ENCODE nucleus transcriptomic data and their Ψ values. circrna cirexon GM12878 HUVEC HeLa-S3 HepG2 K562 chr10: : n/a n/a chr11: : n/a n/a n/a n/a chr12: : n/a n/a n/a n/a chr19: : n/a n/a n/a chr5: : n/a n/a n/a chr19: : n/a n/a n/a n/a chr16: : n/a n/a n/a chr3: : n/a n/a n/a n/a chr5: : n/a n/a 0.25 n/a n/a chr12: : n/a n/a n/a 0.1 n/a chr15: : n/a n/a n/a n/a chr16: : n/a n/a n/a n/a chr16: : n/a n/a n/a n/a chr19: : n/a n/a n/a chr19: : n/a n/a n/a n/a chr2: : n/a n/a n/a n/a chr2: : n/a n/a n/a n/a chr9: : n/a n/a n/a n/a chr9: : n/a n/a n/a chr18: : n/a n/a n/a n/a chr22: : n/a n/a n/a n/a chr22: : n/a n/a n/a n/a 0.5 chr8: : n/a n/a n/a n/a 0.215

41 Supplementary Table 10. Samples of fruit fly used in this study for cirexon/exon detection. Sample Data_amount #BSJs #Cirexons virgin_female_eclosion_1_day_heads 17.6G mated_female_eclosion_1_day_heads 18.2G mated_male_eclosion_1_day_heads 16.9G virgin_female_eclosion_4_day_heads 23.0G mated_female_eclosion_4_day_heads 17.7G mated_male_eclosion_4_day_heads 21.2G virgin_female_eclosion_20_day_heads 16.0G mated_female_eclosion_20_day_heads 16.9G mated_male_eclosion_20_day_heads 19.3G virgin_female_eclosion_4_day_ovaries 19.6G mated_female_eclosion_4_day_ovaries 18.4G mated_male_eclosion_4_day_testes 11.6G mixed_males_and_females_eclosion_1_day_carcass 15.7G mixed_males_and_females_eclosion_4_day_carcass 16.4G mixed_males_and_females_eclosion_20_day_carcass 15.2G mixed_males_and_females_eclosion_1_day_digestive_system 15.9G mixed_males_and_females_eclosion_4_day_digestive_system 16.7G mixed_males_and_females_eclosion_20_day_digestive_system 19.3G adult_midgut 22.8G embryos_0-2_hr_after_egg_laying 23.2G embryos_2-4_hr_after_egg_laying 14.1G embryos_4-6_hr_after_egg_laying 14.5G embryos_6-8_hr_after_egg_laying 13.0G embryos_8-10_hr_after_egg_laying 10.0G embryos_10-12_hr_after_egg_laying 11.2G embryos_12-14_hr_after_egg_laying 19.9G embryos_14-16_hr_after_egg_laying 12.4G embryos_16-18_hr_after_egg_laying 13.1G embryos_18-20_hr_after_egg_laying 23.6G embryos_20-22_hr_after_egg_laying 12.7G embryos_22-24_hr_after_egg_laying 21.1G L3_stage_larvae_dark_blue_gut_PS G L3_stage_larvae_light_blue_gut_PS G L3_stage_larvae_clear_gut_PS_ G third_instar_larvae_wandering_stage_digestive_system 16.6G third_instar_larvae_wandering_stage_cns 30.2G third_instar_larvae_wandering_stage_imaginal_discs 29.4G third_instar_larvae_wandering_stage_salivary_glands 9.3G WPP_salivary_glands 21.0G larval_midgut 41.1G Pupae_WPP_2d_CNS 13.9G S2-DRSC 9.6G S2R 9.0G S1 8.8G

42 ML-DmBG3-c2 9.3G BG3-C2-R2-untreated 12.2G BG3-C2-R2-5h_20E 11.4G BG3-C2-R2-24h_20E 11.6G BG3-C2-R3-5h_20E 12.5G BG3C2_0hr_20E 19.9G BG3C2_1hr_20E 19.8G BG3C2_3hr_20E 9.2G BG3C2_5hr_20E 10.0G BG3C2_7hr_20E 19.3G Kc167-R1-untreated 9.4G Kc167-R2-untreated 11.6G Kc167-R1-5h_20E 9.4G Kc167-R2-5h_20E 9.2G Kc167-R1-24h_20E 12.2G Kc167-R2-24h_20E 9.9G CME_W2 10.9G CME_L1 10.8G