signiﬁcance of piRNA originating outside of transposable elements, if any, is presently unknown. While most reports provide no indications of piRNA and Piwi-family proteins presence outside of the mamma- lian and ﬂy germlines, or outside ﬂy ovarian follicle cells (1,3–5), one study cloned and ampliﬁed several dozens of piRNA-like small RNA (pilRNA) from a variety of mouse tissues (19). More recently, a speciﬁc pilRNA implicated in transcriptional silencing of Ig-like receptors was described in human Natural Killer (NK) cells (20). Finally, thousands of pilRNA, displaying known piRNA sequence features, including presence of 20-O-methyl group at the 30-end, have been reported to be present in Drosophila heads (21). MATERIALS AND METHODS Sample preparation, tissues All rhesus macaque samples were obtained from the Suzhou Experimental Animal Center (Suzhou, China). All tissues were dissected and frozen in liquid nitrogen within 20 min after death. For small RNA library construction, we isolated total RNA from testes, epi- didymis, prostate, seminal vesicles and brain cortex (prefrontal cortex, corresponding to Brodmann area 10) of one adult rhesus macaque individual (9 years 104 days) (see Supplementary Table S6 for sample characteristics). Adult male mice were purchased from the Animal Center of the Chinese Academy of Sciences (Shanghai, China). Experiments were conducted according to a protocol approved by the Institute Animal Care Committee. The protocol conforms to internationally accepted guidelines for the human care and use of labora- tory animals. Mouse epididymis and pancreas samples were obtained from male mice after they were sacriﬁced and the samples were frozen immediately in liquid nitrogen. Total RNA was ﬁrstly isolated using the Trizol (Invitrogen, USA) protocol. In addition, low molecular weight RNA with length ranging from 20 to 40 nt were isolated, ligated to the adapters, ampliﬁed and sequenced following the Small RNA Preparation Protocol (IL, USA), with no modiﬁcations. Sample preparation, speciﬁc cell types Preparation of speciﬁc cell type samples was done independently from the preparation of other tissue samples. Laser Capture Microdissection (LCM) was used to dissect two types of epididymis cells: principal/ basal cells (22,23), which contained pilRNA according to in situ hybridization results and, peritubular tissue, which did not contain pilRNA, from four adult rhesus macaque epididymis samples (Supplementary Table S6). Speciﬁcally, frozen rhesus macaque epididymis tissue was embedded in OCT. Tissue sections (6–8 mm thick) were stained using the Arcturus HistoGene Frozen Section Staining Kit (Molecular Devices) according to the manufacturer’s instructions, with no modiﬁcations. For separating the principal/basal cell and peritubular tissue, Nucleic Acids Research, 2011, Vol. 39, No. 15 6597 the Laser Capture Microdissection System Arcturus, Veritas Microdissection System (Molecular Devices) was used with the following settings: 30–40 mm laser spot size, 88–95 mW power and 2500–3800 ms pulse duration. Groups of cells to be collected were localized and marked under the microscope and then transferred into the provided cap (Arcturus, Molecular Devices). Total RNA was extracted from the cell obtained from LCM, using the RNAqueous -Micro Kit (Ambion) following the manufacturer’s instructions and with no modiﬁca- tions. The Small RNA Preparation Protocol (IL, USA) was applied to the LCM dissected samples with modiﬁca- tions, at only one step, such that prior to low molecular weight RNA isolation, the total RNA from four rhesus individuals aged between 8 and 10 years was combined in equal amounts. All small RNA sequencing data (ﬁve macaque tissue samples, two macaque epididymis LCM-dissected samples and one mouse epididymis sample) are deposited to GEO under accession number GSE27369. Small RNA mapping We mapped the small RNA sequencing data as described elsewhere (24). Brieﬂy, for each rhesus macaque sequencing data set, all multiple reads representing one sequence were collapsed and the adapter sequence at the 30-end of the sequence reads was trimmed using the custom trimming procedure. The resulting unique sequences (sequence tags) were mapped to the rhesus macaque genome (MGSC Merged/rheMac2) using Bowtie (25). We allowed one mismatch for the sequences with length >25 nt and required perfect match for the shorter sequences. According to these criteria, 58–69% of all reads ( 7–9 million reads) could be mapped to the rhesus macaque genome of each sample (Supplementary Table S7). We downloaded 57 mouse small RNA sequencing data sets, from GSE12757 (7), GSE17319 (26), GSE19172 (16), GSE7414 (10), GSE16023 (27) and GSE21630 (28), and 60 Drosophila melanogaster small RNA sequencing data sets from GSE11624 (29), GSE12462 (30), GSE11019 (21), GSE10794 (31), GSE11086 (32), GSE7448 (33), GSE18806 (30), GSE6734 (1) and GSE15378 (11) (Supplementary Table S13). Downloaded mouse data and small RNA sequences from mouse epididymis, as well as downloaded ﬂy data, were processed and mapped to the mouse genome (NCBI37/mm9) and the D. melanogaster genome (BDGP R5/dm3), respectively, using the above described procedure. Rhesus macaque genome annotation The rhesus macaque genome (MGSC Merged/rheMac2) was downloaded from UCSC (34). The rhesus macaque transposon/repeat annotation was downloaded from UCSC rhesus RepeatMasker track (34). We classiﬁed LINE, SINE, LTR and DNA as ‘transposons’ and the rest of the repetitive sequence types, such as simple repeats, as ‘other repeats’ The rhesus macaque protein-coding genes and non-coding RNA (ncRNA) annotation was based on