complementary to small RNA sequences highly expressed in two piRNA clusters in macaque testes (Figure 3; Supplementary Table S10; ‘Materials and Methods’ section). In mice, one of the orthologous clusters was shown to generate large numbers of piRNA in adults, but not in prenatal or 10 day-old animals (7,10) (Supplementary Figure S8 and Supplementary Table S11). Accordingly, we found few small RNA originating from this genomic region in immature rhesus macaque testes (Supplementary Figure S8 and Supplementary Table S11). For all LNA probes, we observed strong in situ hybridization patterns in adult macaque testes (Figure 4A and Supplementary Figure S9). Notably, we also observed clear hybridization signals in the four somatic tissues, most of them localized within speciﬁc cell types (Figures 4A and Supplementary Figures S10–S16). In the epididymis and the prostate, the hybrid- ization signal was located in the principal and basal cells of epithelia surrounding the ducts, but not in the peritubular tissue or spermatozoa (Supplementary Figures S10–S12). In the cerebral cortex, the hybridization signal showed a pattern resembling a neuronal body distribution (Supplementary Figure S14). These hybrid- ization patterns were consistent among different ‘anti-pilRNA’ LNA probes and were reproduced in another adult macaque individual (Supplementary Figures S15 and 16). Presence of piRNA requires expression of Piwi-family proteins. In the absence of suitable antibodies, we checked mRNA expression of the four macaque PIWI proteins, (PIWIL1-4), by in situ hybridization. We found that PIWIL mRNA were expressed both in testes and in somatic tissues. Most importantly, in all cases PIWIL mRNA expression co-localized with hybridization signals from ‘anti-pilRNA’ LNA probes (Figure 4A). While in testes PIWIL1 mRNA had the strongest signal, in somatic tissues PIWIL2 and PIWIL4 mRNA signals were the strongest ones. Although not quantitative, this observation is consistent with previous reports of PIWIL4 mRNA expression across human somatic tissues (47) and indicates that pilRNA might be associated with a subset of PIWI proteins. The LNA-probes used in our study cannot discriminate between small and long transcripts. To test whether hybridization signal comes from pilRNA, rather than long transcripts, we isolated and sequenced small RNA from cells with and without LNA hybridization signal. Cells stained after LNA-probes hybridization should contain pilRNA, while unstained cells should not. Using laser capture microdissection (LCM), we dissected princi- pal and basal cells that had a clear LNA hybridiza- tion signal and peritubular tissue that had no signal, from epididymis of four adult rhesus macaques (Supplementary Table S6; Figure 4A and B; Supplementary Figure S11). We indeed found that the principal and basal cells contained 30 times more pilRNA reads than the peritubular tissue. In contrast, expression of other small RNA did not differ between these cell types (Figure 4C). Furthermore, expression of pilRNA in the principal/basal cells correlated well with expression of pilRNA found in the total epididymis Nucleic Acids Research, 2011, Vol. 39, No. 15 6605 (R = 0.85, P < 2.2 e-16). The residual presence of pilRNA in the peritubular tissue, detected by sequencing, might be caused by their incomplete separation from the epithelial cells in the LCM procedure. Based on these results, we can conclude that principal/basal cells of rhesus macaque epididymis indeed contained pilRNA, while the peritubular tissue did not. This result could not be explained by spermatozoa contamination, as LCM-dissected cells did not contain detectable spermatozoa (Figure 4B) and, according to in situ hybrid- ization results, spermatozoa did not contain detectable levels of pilRNA (Figure 4A and Supplementary Figure S11). A previous study, which used the northern blot hybrid- ization technique with speciﬁc probes, did not detect presence of piRNA in mouse epididymis (5). To test weather piRNA are indeed absent in this tissue, we sequenced small RNA from mouse epididymis, following the same procedure (‘Materials and Methods’ section). As in macaques, mouse epididymis contained small RNA with length 24–32 nt that formed pilRNA clusters. These pilRNA clusters largely overlapped with piRNA clusters identiﬁed in mouse testes (96%, P < 0.0001) and contained sequences showing a clear ping–ping ampliﬁ- cation signature, as well as other piRNA features (Supplementary Table S4 and ‘Materials and Methods’ section). Furthermore, epididymis pilRNA coverage across the orthologous clusters was largely conserved between mice and macaques (R = 0.57, P < 10 13). Thus, presence of pilRNA in epididymis could be observed in both mice and macaques. Why, then, were mouse epididymal pilRNA not detected previously? Using our sequence data, we tested the abundance of small RNA complementary to the four piRNA-speciﬁc probes used in the previous studies in order to detect piRNA expression in mouse epididymis and ovaries (4,5). We found that levels of these speciﬁc small RNA were, on average, 75-fold lower in ovaries and epididymis compared to testes (Supplementary Figure S17; Supplementary Table S12; ‘Materials and Methods’ section). Thus, it is possible that the chosen probes did not provide enough sensitivity. Indeed, while no hybridization signal using these probes was detected in both epididymis and ovaries (4,5), piRNA presence in mouse ovaries has since been described (7,28,48,49). Taken together, our results demonstrate that small RNA sharing sequence features of piRNA are widespread among somatic tissues in ﬂies, mice and macaques. Notably, in all three species, pilRNA found in somatic tissues, with possible exception of several mouse cell types, resembled germline piRNA, rather than piRNA present in ﬂy ovarian somatic cells. Even though our study did not test association between pilRNA and Piwi proteins, presence of pilRNA across animal somatic tissues is intriguing. In some tissues, this presence was quite substantial: in macaque epididymis pilRNA constituted 15% of the total mapped reads and in mouse pancreas 3.5%. While 3.5% is not a large proportion, it was greater than the proportion of total reads (3%) occupied by piRNA in mouse ovaries. Furthermore, our in situ hybridization results indicated that pilRNA