Nucleic Acids Research, 2011, Vol. 39, No. 15 6601 Figure 3. piRNA and pilRNA features in rhesus macaque tissues. (A) Length distribution of all mapped sequence reads in ﬁve adult rhesus macaque tissues and adult mouse testes. The labels indicate: R.Te, macaque testes; R.Ep, macaque epididymis; R.Pr, macaque prostate; R.Sv, macaque seminal vesicles; R. Co, macaque cerebral cortex; M.Te.All, mouse total testes (26); M.Te.ML, mouse testes Mili-immunoprecipitation (16); M.Te.MW, mouse testes Miwi-immunoprecipitation (16) (B) Length distribution of all mapped sequence tags (sequence reads after collapsing identical sequences). (C and D) Length distributions of sequence reads and tags, excluding reads mapped to known ncRNA, respectively. Note that 50 uridine bias is one known property of primary piRNA. (E) Proportions of complementary small RNA reads (24–32 nt) at different offsets. Excess of complementary reads at offset equal 10 nt indicates the ping-pong model signature. (F) The stand (S) and the 50 uridine (U) bias of sequence reads’ clusters based on 24–32 nt long sequences. (G) Reads coverage of an example piRNA with high coverage in adult macaque testes. Shown are all reads with length 24–32 nt, excluding reads mapped to known ncRNA. The y-axis shows log2-transformed nucleotide coverage normalized by the total number of mapped reads within in a given sample. The bottom of the panel shows annotation of the corresponding rhesus macaque genomic region, dark green—SINE and location of one of the ‘anti-pilRNA’ LNA probes (piRNAa)—arrow. Note that the genomic region orthologous to the macaque small RNA cluster shown in Figure 3G is inverted in the mouse genome. To align the mouse and macaque genomic regions, mouse cluster is shown in reverse orientation with respect to the macaque cluster. RNA clusters found in mouse and rhesus macaque epididymis. In comparison of pilRNA cluster expression between stained and unstained cells dissected from rhesus macaque epididymis by LCM, we took a union of small RNA clusters identiﬁed in these two samples. The control clusters were required to contain at least one uniquely mapped sequence and show <40% 50 U-bias in the two samples. Comparison of rhesus macaque testes small RNA clusters with human and mouse known piRNA To compare rhesus macaque testes, small RNA clusters with known human testes piRNA clusters, we downloaded human piRNA cluster coordinates from (3) and mapped rhesus macaque small RNA clusters to the human genome using reciprocal liftOver. To check how many rhesus macaque small RNA clusters (piRNA and/or pilRNA) could be supported by known mouse piRNA, we mapped rhesus macaque clusters to the mouse genome (mm9) using reciprocal liftOver. Then we checked mouse piRNA density within each liftovered cluster region using piRNA sequences from published Piwi-family proteins immunoprecipitation studies (7,10,16,26,27). To simulate mouse piRNA density expected by chance, we randomly chose start coordinates of the clusters, while keeping the length consistent, 1000 times. Measure the expression level of four previous piRNA candidates using Deep sequencing data We measured expression levels of four speciﬁc piRNA, used as piRNA markers across tissues in previous studies (4,5), in deep sequencing data sets from mouse testes, ovary (28) and epididymis. Speciﬁcally, for each piRNA candidate, small RNA sequence reads that mapped within 50 nt upstream and downstream of the genomic location of the piRNA sequences were summed (Supplementary Table S12). Expression patterns of piRNA-like molecules in ﬁve rhesus macaque tissues using in situ hybridizations We designed three LNA-probes (Supplementary Table S10) corresponding to highly expressed small RNA sequences. Hybridizations were performed as