acids 826–845) and mouse anti-actin antibody (Sigma) were used for immunoblotting as described in (42). Ethics statement All rhesus macaques and mice used in this study suffered sudden deaths for reasons other than their participation in this study and without any relation to the tissue used. Biomedical Research Ethics Committee of Shanghai Institutes for Biological Sciences completed the review of the use and care of the animals in the research project (approval ID: ER-SIBS-260802 P). RESULTS AND DISCUSSION Small RNA in ﬂy tissues To assess presence of piRNA-like small RNA in D. melanogaster tissues, we analyzed publicly available small RNA data sets from embryo, ovary, head, imaginal discs and OSS cells generated using high-throughput sequencing (11,16,21,29–33). Embryo, ovary and OSS cells were previously shown to contain piRNA (1,11,16,29,32). Indeed, we observed large numbers of sequences with length 24–32 nt, an interval corresponding to the piRNA length, in small RNA data from these three tissues (1,11,16,29). Besides length, these small RNA showed other known properties of ﬂy piRNA, such as 50 uridine bias, clustering along the genome and preferential localization in the antisense repeat regions (54–65%) (Figure 1A–D, F and G). Further, small RNA present in embryo and ovaries showed clear signature of the ping-pong biogenesis model and low piRNA cluster strand speciﬁcity (Figure 1E and F; Supplementary Figures S1 and S2). In contrast, in agreement with previous reports (11), OSS cells did not show any detect- able ping-pong model signature and largely contained strand-speciﬁc piRNA clusters (Figure 1E and F; Supplementary Figures S1 and S2). Thus, overall analysis of high-throughput sequencing data from ﬂy embryo, ovary and OSS cells has allowed us to ‘rediscover’ piRNA in these tissues by identifying small RNA that display all previously reported piRNA sequence features. Notably, applying the same analysis to the ﬂy head and imaginal disc data sets, we also identiﬁed a distinct fraction of small RNA, with length distribution of 24–32 nt, displaying piRNA sequence features. Presence of these piRNA-like small RNA (pilRNA) was not affected by exclusion of sequences originating from known non-coding RNA (ncRNA) (Figure 1C and D). Further, similar to piRNA, head and imaginal disc pilRNA showed 50 uridine bias, clustering along the genome and preferential localization in the antisense repeat regions (Figure 1 and Supplementary Tables S1–S3). More than 55% of pilRNA clusters found in both head and imaginal discs overlapped with previously reported ovarian piRNA clusters, while <10% would be expected to overlap by chance (P < 0.0001). Further, pilRNA cluster coverage in ﬂy head and imaginal discs correlated well with piRNA cluster coverage in ﬂy ovaries (R > 0.7, P < 0.0001). Nucleic Acids Research, 2011, Vol. 39, No. 15 6603 Both head and imaginal disc pilRNA showed a distinct ping-pong model signature and low cluster strand speciﬁ- city, characteristic to ﬂy germline piRNA (Figure 1E and F; Supplementary Figures S1 and S2; Supplementary Tables S1–3). Further, as is the case for ﬂy germline piRNA (11), head and imaginal disc pilRNA were enriched in 42AB genomic region and were under- represented in 20AB/ﬂamenco locus (Figure 1G and Supplementary Table S2). Finally, in agreement with previous observations (21), the proportion of pilRNA detected in ﬂy heads increased substantially after enrich- ment for small RNA containing 20-O-methyl group at its 30-end, a known piRNA sequence feature (43–46), during small RNA library preparation. This enrichment could be reproduced in our analysis in three independent small RNA data sets (21,30) (Supplementary Figure S3). Thus, our analysis conﬁrmed previously reported presence of pilRNA in ﬂy heads and showed presence of pilRNA in ﬂy imaginal discs. In both tissues, the propor- tion of pilRNA, deﬁned as small RNA with length between 24 and 32 nt after exclusion of known ncRNA, was lower than in tissues previously shown to contain piRNA: embryo, ovary or OSS cells. Speciﬁcally, average proportions of pilRNA among all mapped sequences were 2 and 14% in heads and imaginal discs and 44, 34 and 59% in embryo, ovary and OSS cells, respectively. In the ﬂy head samples, this proportion increased to an average of 10% after enrichment for RNA containing 20-O-methyl group. Despite smaller numbers, however, pilRNA present in ﬂy heads and imaginal discs showed all known sequence properties of ﬂy germline piRNA, as well as signiﬁcant overlap of cluster locations and signiﬁcant correlation of the cluster coverage. It was surprising that pilRNA present in ﬂy somatic tissues did not resemble piRNA found in ovarian follicle cells, another somatic tissue, but showed all features of piRNA found in the germline. Without proof of direct association between these small RNA and Piwi proteins, however, they cannot be classiﬁed as piRNA. Still, the fact that substantial numbers of small RNA displaying piRNA features are present in ﬂy somatic tissues might, by itself, be worthwhile noting. Small RNA in mouse tissues To investigate pilRNA presence in mammalian tissues, we took advantage of a published data set, which contains small RNA sequences from 11 mouse tissues and 31 immune system cell types (28). In agreement with previous reports (28), in these data, we detected abundant piRNA presence in testes and moderate numbers of piRNA in ovaries. In addition to germline, we found small RNA showing distinct sequence features of mammalian piRNA in 17 out of the 40 mouse somatic tissues and cell types (‘Materials and Methods’ section, Supplementary Table S4 and S5). Speciﬁcally, in addition to universal piRNA features, such as length distribution between 24 and 32 nt, 50 uridine bias and clustering along the genome, we observed other sequence features typical to the mammalian piRNA: strand speciﬁ- city of the clusters, under-representation in repetitive