6600 Nucleic Acids Research, 2011, Vol. 39, No. 15 Figure 2. piRNA and pilRNA features in mouse tissues. (A) Length distribution of all mapped sequence reads in three adult mouse tissues: Te, testes; Ov, ovary; Pa, pancreas. (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 a bidirectional piRNA cluster previously identiﬁed in mouse testes (4) in three mouse tissues. 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. (H) Genomic context of piRNA clusters found in mouse testes at different age, from embryonic stages, pre-pachytene (10 dpp) and pachytene (18 dpp) stages to adults, based on data from (7,10,16,26,27) (‘Materials and Methods’ section). The labels indicate the age of the samples in days post-conception (dpc), days post-partum (dpp) or weeks (w) and piRNA origin: testes tissue (no label) or immunoprecipitations of Mili (Ml), Miwi2 (Mw2) or Miwi (Mw) proteins. For a full sample description, see Supplementary Table S13. (I) Genomic context of pilRNA clusters in adult mouse tissues: GC-B, germinal center B cells; Ma-B, mature B cells; Pl, plasma cells; FH-T, follicular helper T cells; NK, natural killer cells; MP, macrophages; MDD, macrophages-derived dendritic cells; Ne, neutrophils; HP, hem- atopoietic progenitor cells; Ov, ovaries; Pa, pancreas; Te, testes (28). above-mentioned criteria, we classiﬁed the tissue as showing the ping-pong ampliﬁcation model signature. In addition, we investigated the ping-pong signature using the method described in (14). This approaches produced similar results (Supplementary Figure S2). To estimate pilRNA strand speciﬁcity, we ﬁrst calculated total pilRNA reads from each strand of one cluster and, further, calculated the ratio between the reads from one strand that has more pilRNA inside, to total pilRNA reads of the cluster. To investigate the genomic context, pilRNA located within clusters were classiﬁed according to their genomic localization: repeat regions, exon regions (including 50-UTR, coding region and 30-UTR), intron regions and no annotation regions. Annotation was done hierarchical- ly in the listed order. A small RNA was assigned to a given annotation category if at least 1 nt mapped within the corresponding genomic region. The relative percentage of small RNA in each annotation category was compared to the percentage occupied by each category in the rhesus macaque genome according to the above-mentioned hierarchical order, using a binomial test. We note that because repeat regions had the highest annotation priority, our results might be biased toward over-annotation of small RNA within repeat regions. To measure four previous TE master loci activity (11) in ﬂy OSS, embryo, ovary, head and imaginal discs, we collected pilRNA mapped within each TE master loci, and used both uniquely mapped reads and weighted reads (reads divided by mapped loci number) to estimate the activities (Figure 1G and Supplementary Table S2). Small RNA cluster expression correlation To calculate the correlation in the expression of small RNA (piRNA and pilRNA) clusters between rhesus macaque testes and rhesus macaque somatic tissues, we deﬁned cluster loci based on small RNA found in rhesus macaque testes. All small RNA clusters identiﬁed independently in somatic tissues overlapped with clusters with small RNA clusters found in testes. To calculate the correlation in the expression of small RNA clusters between ﬂy ovaries or mouse testes and other ﬂy or mouse tissues, respectively, for each comparison, we considered the union of small RNA clusters found in the two tissues. In comparison of small RNA cluster expres- sion between mouse and rhesus macaque epididymis, reciprocal liftOver was used to ﬁnd corresponding small RNA cluster regions in the other species. The expression correlation was calculated based on the union of small