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Single-base resolution DNA methylomes have been accomplished for both Arabidopsis and human cells which have high genome methylation levels by Illumina ultra-high-throughput bisulfite sequencing technology (MethylC-Seq). Here by combining MethylC-Seq and biological replicate strategies we generated single-base resolution methylome for the silkworm which has low genome methylation levels like other insects. Our conservative estimation showed that methylcytosines (mCs) accout for about 0.11% of genomic cytosines, exclusively in CG context. The CG methylation is significantly enriched in gene bodies and positively correlated with gene expression levels, suggesting its positive role in gene transcription in silkworms. However, the well-documented functions of methylation on promoters and rDNAs in plants and mammals do not seem to have effects in insects. Methylated genes are enriched in functions involved in cellular metabolism and biosynthesis. Small RNA (smRNA) loci are also significantly enriched in gene bodies, and moreover, the smRNA loci and the predicted target sites of microRNA have high level of CG methylation, indicating functional involvement of smRNAs in the genic methylation This first methylome for silkworms provides a foundation for further studies on the epigenetic gene regulation of silkworms’ or even insects’ gene methylation. Each silk gland of 5th instar larvae of two individuals (called Biological Replicate 1 and 2, respectively) of the silkworm (Bombyx mori) strain Dazao was ground into powder in liquid nitrogen. Half of the powder from each silk gland was used to extract total DNAs using DNeasy Blood & Tissue Kit (Qiagen) and another half was used to extract total RNAs using RNeasy Mini Kit (Qiagen). We sequenced bisulfite-treated total DNA extracted from the silk glands of the two individuals, using Illumina Ultra-High-Throughput Sequencing, generating the Single-Base Resolution Methylomes. To reveal functional consequences of gene body methylation, we generated expression profiles for the two individuals’ silk glands using Digital Gene Expression tag profiling (DGE) technology, which combines classic SAGE (Serial Analysis of Gene Expression) and Illumina ultra-high-throughput sequencing technology.

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