An intergenic pachytene piRNA cluster MIWI-immunoprecipitated small RNA (>28 nt) is a guide piRNA that targets a cleavage sequence (−11 nt to +10 nt of cleavage site) if it first overlaps the 5′ end of the corresponding 5′RACE tag in an antisense manner by exactly 10 nt, allowing for a mismatch in piRNA nucleotide 1. In addition, it must have perfect complementarity in the primary seed (piRNA nucleotides 2–11) and tolerate at most four mismatches in the secondary seed (piRNA nucleotides 12–21). The complementarity cutoff and seed definitions are based on criteria that allow mouse Bbs5 and Gm11837 to be targeted by only Hu6 human piRNAs but not a single native mouse piRNA and also take into consideration previous in vitro small RNA-based cleavage assays (Reuter et al. 2011 (link); Nakanishi et al. 2012 (link)).
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Pachytene piRNA-Guided Cleavage Criteria
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Other organizations : Cold Spring Harbor Laboratory, Howard Hughes Medical Institute, Cancer Research UK, University of Cambridge, University of Turku
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Variable analysis
independent variables
- Presence of a guide piRNA that overlaps the 5' end of the corresponding 5'RACE tag in an antisense manner by exactly 10 nt, allowing for a mismatch in piRNA nucleotide 1
- Presence of perfect complementarity in the primary seed (piRNA nucleotides 2–11) and at most four mismatches in the secondary seed (piRNA nucleotides 12–21)
- Cleavage of a target sequence (-11 nt to +10 nt of cleavage site)
- Criteria that allow mouse Bbs5 and Gm11837 to be targeted by only Hu6 human piRNAs but not a single native mouse piRNA
- Previous in vitro small RNA-based cleavage assays (Reuter et al. 2011; Nakanishi et al. 2012)
- Positive control: Hu6 human piRNAs targeting mouse Bbs5 and Gm11837
- Negative control: Native mouse piRNAs not targeting mouse Bbs5 and Gm11837
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