The antibody used to immunoprecipitate (IP) piRNAs is anti-PIWIL1 (Abcam, ab12337), originally raised against the human PIWIL1 protein C-terminal peptide. Immunoprecipitation, Illumina library preparation, deep sequencing, and bioinformatic analysis of Piwi-associated small RNAs from the sexual progeny of both WT and backcrossed cells followed our published protocol.19 (link) Small RNA sequences were mapped onto the micronuclear region containing contig11396-TEBPβ with the gmapper command in the SHRiMP software package,62 (link) setting the threshold score to 80% of the maximum possible score using the ‘-h 80%' flag. To ensure comparable depth between control and fusion libraries, we subsampled to equivalent sequencing depth of 35 million reads per library.
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Profiling piRNA-Piwi Interactions
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- Anti-PIWIL1 (Abcam, ab12337) antibody used to immunoprecipitate (IP) piRNAs
- Piwi-associated small RNAs from the sexual progeny of both WT and backcrossed cells
- Immunoprecipitation, Illumina library preparation, deep sequencing, and bioinformatic analysis of Piwi-associated small RNAs followed the published protocol
- Small RNA sequences were mapped onto the micronuclear region containing contig11396-TEBPβ with the gmapper command in the SHRiMP software package, setting the threshold score to 80% of the maximum possible score using the '-h 80%' flag
- Sequencing depth was subsampled to 35 million reads per library to ensure comparable depth between control and fusion libraries
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