Oey et al. BMC Genomics 2011, 12:315 http://www.biomedcentral.com/1471-2164/12/315 both were used to estimate the typical ratio of pr19RNAs relative to s-piRNAs genome-wide. Miwi and Mili IP analyses The IP datasets were mapped to the LINE and IAP con- sensus sequences as well as miRNAs (miRBase v.16) [21], tRNAs [22], snoRNAs [23,24] and the mitochon- drial genome (NCBI37/mm9). The number of mapped reads were then normalised to the number of genomi- cally mappable reads in each library as reads per million. Strand-specific real-time quantitative reverse transcription PCR RNA was extracted from 4-month old C57BL/6 mouse testes with Trizol (Invitrogen), 2 μg was treated with DNase I (New England Biolabs) and used for reverse transcription (RT). RT was carried out using Superscript III (Invitrogen) according to manufacturer’s instructions, apart from the following changes; MgCl2 was added to a final concentration of 5 mM and the RT step was per- formed at 50°C. Two strand-specific RT reactions were carried out against each strand of the piRNA cluster using the forward and reverse primers to prime the first-strand synthesis (Target1-forward: GCCTATTGCTCCTTGGACTG, Target1-reverse: AGC- TAGCTGAAATCGGATGG, Target2-forward: AGGAAAAAGGTTGCCGATCT, Target2-reverse: CTCTTGAAAGGGCTTCTTGC. GAPDH was reverse transcribed in parallel and used to normalise between the reactions (GAPDH-forward: TCCAGAGACAGCCG- CATCT, GAPDH-reverse: ACACCGACCTTCAC- CATTTTG). Real-time quantitative RT PCR (qRT-PCR) was performed in triplicate using Platinum SYBR Green qPCR mix (Invitrogen) on a Corbett Rotorgene qPCR machine at the default settings. Differences in expres- sion between strands were estimated using the 2-ΔΔCT method [25]. Results Small RNAs derived from IAPs in testis It is known that p-piRNAs target retrotransposons such as IAPs and LINEs and facilitate cleavage and repression of these genomic parasites in the testes [9,11]. In order to investigate small RNAs with homology to such ele- ments we first mapped small RNAs from a publicly available spermatogenic tubule dataset [7] to the full genomic complement of mouse IAP repeats using strin- gent alignment parameters. This provided us with a database of IAP-derived reads that were then remapped with less stringent parameters to an IAP consensus sequence. As expected, many of the IAP-derived reads were ~24-30 nt long with sequence features suggesting they were piRNAs (Figure 1A and 1B). However, we also noticed a similarly abundant group of precisely 19 Page 3 of 10 nt long RNAs (Figure 1A). Both the 19mers and the piRNAs were found to map in similar numbers to the sense and the antisense strands of the IAP repeats, a distribution that has been shown previously for the piR- NAs and that may be explained by widespread sense and antisense transcription of these elements [11,26]. The 19mers mapped throughout the full length of the IAP repeat, including the long terminal repeat (LTR) regions (Figure 1C). We aligned the 19mers in an effort to identify overrepresented internal nucleotides since many functional small RNAs are found to have such internal preferences [27,28]. No internal patterns emerged and we extended the alignments both upstream and downstream using the IAP reference sequence as a proxy for the original IAP transcripts. These alignments revealed a strong preference for A 10 nt downstream of most 19mers (Figure 1B). Out of the 9,648 19mers that were mapped to the sense strand of the IAP repeat, which has the strongest downstream sequence bias, 7,991 (82%) have an A 10nt downstream, significantly more than the ~25% one would expect by chance. These RNAs are reminiscent of a 19nt long small RNA class recently characterised by Berninger et al. [12] using non-repetitive small RNA deep sequencing reads. Because s-piRNAs have a known preference for A at position 10, it is likely that the 5’ ends of the 19mers were derived from a cleavage event 19 nt upstream of the 5’ termini of these s-piRNAs. This suggested that many of the 19mers would be flanked by s-piRNAs at their 3’ end and overlap with opposite sense p-piRNAs. We tested this hypothesis by tallying the 5’ termini of same-sense s-piRNAs and opposite-sense p-piRNAs relative to the 5’ ends of the 19mers. As predicted, we found this to be the case (Figure 1D and Additional file 1 Figure S1A). This positional relationship between the 19mers and piRNAs was also described by Berninger et al. [12] using non-repetitive reads and it mirrors the relationship between p-piRNAs and s-piRNAs previously described by others [9,11]. The correlation in the distri- bution of 19mers relative to p-piRNAs and and s-piR- NAs is also evident when plotting the density of these along the IAP reference sequence where the 19mers are often found to overlap with same-sense s-piRNAs and opposite sense p-piRNAs (Figure 1C). Out of the 2,743 reads identified as s-piRNAs (RNAs 24-30nt in length with A in position 10 and absence of U in position 1) mapped to the sense-strand of the IAP consensus sequence, 1,931 (70%) were found immediately adjacent to a 19mer (configuration illustrated in Figure 1D). Similarly, out of 4,985 p-piRNAs (RNAs 24-30nt in length with U in position 1 and absence of A in position 10) mapped in antisense to the IAP consensus sequence, 4,434 (89%) were found to have 5’ termini located pre- cisely 10 bases downstream of the 3’ terminus of a