6604 Nucleic Acids Research, 2011, Vol. 39, No. 15 element regions and compatibility with the ping-pong piRNA biogenesis model (‘Materials and Methods’ section, Supplementary Figure S2 and Supplementary Table S4 and S5). While in some tissues not all of these features could be detected due to relatively small pilRNA cluster numbers, they were apparent in tissues with sub- stantial pilRNA cluster numbers, such as in the pancreas (Figures 2 and Supplementary Figure S2; Supplementary Table S4 and S5). With respect to genomic context and ping-pong ampliﬁcation features of identiﬁed pilRNA, mouse tissues could be separated into two types: resembling the germline and resembling the OSS. Pancreas, hematopoi- etic progenitor cells, neutrophils, macrophages and macrophage-derived dendritic cells contained pilRNA clusters preferentially located in unannotated genomic regions and vastly overlapping with piRNA clusters identiﬁed in mouse testes using piRNA co-precipitating with PIWI proteins (average overlap 97.2%) (Figure 2I and Supplementary Table S4). In contrast, pilRNA found in germinal center B cells, mature B cells, plasma cells, follicular helper T cells and NK cells showed bias toward the 30-UTR sense strand regions and overlapped only weakly with piRNA clusters found in mouse testes (average overlap 21.4%) (Figure 2I and Supplementary Table S4). The piRNA bias toward the 30-UTRs was previously described for mouse pre-pachytene testes (Figure 2H) and Drosophila OSS cells (Figure 1G) (10,16). Notably, in both cases only one of the Piwi-family proteins (Mili or Piwi, respectively) was expressed (10,16). Based on these observations, we could speculate that the difference between the two pilRNA types could be caused by expression of different sets of Piwi-family proteins in these tissues. Interestingly, pilRNA presence in B cells appeared to require an intact activation-induced cell death mechanism, as none—or very few—pilRNA could be found in germinal center and mature B cells, in the BclXL mouse strain (Supplementary Table S4). While apoptosis might lead to generation of small RNA through mRNA degradation, experimental procedure used for small RNA library preparation should not result in biased pilRNA distribution along transcripts in germinal center and mature B cells in the wild type mice. Our observation, that pilRNA, found in these cells show preferential local- ization in 30-UTR is not compatible with a simple mRNA degradation scenario (‘Materials and Methods’ section and Supplementary Table S4). Thus, our analysis of published small RNA sequencing data showed widespread presence of pilRNA across mouse somatic tissues. In most of these tissues, the total proportions of pilRNA were small and the proportions of pilRNA that fell into clusters were yet smaller: 0.6% on average. In pancreas, however, pilRNA presence was substantial 3.5% of the total mapped reads. Notably, the proportion of total reads occupied by piRNA in mouse ovaries, deﬁned using the same criteria, was 3%. Furthermore, 76% of all pancreas pilRNA reads mapped within pilRNA clusters. Similarly, 82 and 83% of piRNA reads mapped within clusters in ovaries and testis, respect- ively. Importantly, western blot results conﬁrmed Miwi protein expression in mouse pancreas (Supplementary Figure S4) Again, as in case of ﬂy pilRNA, mouse pilRNA found in our study cannot be classiﬁed as piRNA until their association with Piwi proteins is shown. Still, substantial presence of pilRNA in some somatic tissues, such as in the pancreas, might indicate their potential functional importance. Small RNA in rhesus macaque tissues We next tested for presence of pilRNA in somatic tissues in a mammalian species closely related to humans—rhesus macaque. To do so, we sequenced small RNA with a size range of 20–40 nt in adult rhesus macaque testes, epididy- mis, prostate, seminal vesicles and cerebral cortex (prefrontal cortex, corresponding to Brodmann area 10) using Illumina sequencing platform (‘Materials and Methods’ section and Supplementary Table S6). For each tissue, we obtained 10 266 594—13 972 125 reads, 58–69% of which could be mapped to the rhesus macaque genome (‘Materials and Methods’ section and Supplementary Table S7). In all tissues except testes, the majority of reads (75–95%) were 21–23 nt in length and mapped within the annotated miRNA precursor regions (‘Materials and Methods’ section). In testes, in agreement with the previous studies (3,26), the majority of the reads (92%) had length of 24–32 nt, corresponding to the length of piRNA (Supplementary Figure S5) and showed all other known piRNA sequence features (‘Materials and Methods’ section; Supplementary Tables S8 and S9; Figure 3). Expression of small RNA found in macaque testes overlapped well with expression of mouse piRNA identiﬁed by co-precipitation with Piwi proteins (7,10,16): out of 1548 macaque clusters with orthologs in the mouse genome, 71% contain 5 or more mouse piRNA, while <8% would be expected by chance (P < 0.0001). Similarly, 92% of the annotated human piRNA clusters (3) overlap with macaque clusters. Taken together, these features indicate that the vast majority of analyzed small RNA sequences with length distribution between 24 and 32 nt correspond to genuine testes piRNA. Unexpectedly, in all other macaque tissues studied, we found small RNA with the same sequence properties. Such properties included length distribution, 50 uridine bias, genomic localization and clustering, cluster strand speciﬁcity and ping-pong model mechanism features (Figure 3; Supplementary Figures S2 and S6; Supplementary Tables S8 and S9). Compared to testes, these pilRNA occupy a much smaller proportion of all mapped sequence reads: 1–15% versus 92% in testes (Supplementary Table S8). Consequently, only a few pilRNA clusters (58–248) can be identiﬁed in somatic tissues (Supplementary Tables S8 and S9). All pilRNA clusters found in somatic tissues overlapped with macaque testes’ piRNA clusters and cluster read coverage correlated well among tissues (R > 0.90, P < 10 15) (Supplementary Figure S7). To further investigate pilRNA expression in somatic tissues, we designed ‘anti-pilRNA’ LNA probes