Characterization of <i>Bombyx</i> somatic piRNAs.

(A) The length distribution of small RNAs in female tissues from fifth instar larvae. The small RNA libraries of WT and BmAgo3 KO were prepared from 3–4 and 4–8 fifth instar larvae, respectively. Small RNAs around 27 nucleotides long in the ovaries (OV), fat body (FB), and wing discs (WD) disappeared in BmAgo3 KO. The y-axis indicates normalized reads per million (RPM). (B) First base bias of small RNAs of around 27 nucleotides in length. The percentages of the first nucleotides of the small RNAs shown in (A) were calculated. (C) Mapping rate of each tissue small RNA library to transposons. (D) Investigation of ping-pong signature. piRNAs amplified by ping-pong have overlapping pairs of 10 bases. (E) piRNA orientation and 1U/10A strand bias for each transposon. For each transposon, the 1U bias and 10A bias for each strand were sorted by the difference between sense and antisense percentages of 1U piRNAs in WT ovaries. (F) MAplot for mRNA expression levels using transposons and silkworm gene models. Total RNA used for RNA-seq analysis was the same as that used for small RNA-seq analysis. The transcripts per million (TPM) of female tissues from WT and BmAgo3 KO were used to calculate the difference in M:log2(TPM) and the mean value of A:log2(TPM). The axes show: A (x-axis) = (log2(TPM in BmAgo3 KO) + log2(TPM in WT))/2. M (y-axis) = log2(TPM in BmAgo3 KO)–log2(TPM in WT). (G) Venn diagram showing the inclusion relationship of transposons producing more than a certain amount of piRNA (normalized RPM > 4). piRNAs are produced from 1011 transposons out of 1811 B. mori transposons examined. Ovarian piRNAs are produced from 1008 transposons, of which ovary-specific piRNAs are produced from 591 transposons. Non-gonadal somatic piRNAs are produced from 412 and 208 transposons in wing discs and fat body, respectively. Common piRNAs in ovary and wing discs are produced from 412 transposons. Common piRNAs in ovary and fat body are produced from 205 transposons. Common piRNAs in the three tissues are produced from 200 transposons. Fat body-specific piRNAs are produced from 3 transposons.