Transcriptome-scale analysis for RNAs of medium length, designated as melRNA-seq

More than one million copies of short interspersed elements (SINEs), a class of retrotransposons, are present in the mammalian genomes, particularly within gene-rich genomic regions. Evidence has accumulated that ancient SINE sequences have acquired new binding sites for transcription factors (TFs) through multiple mutations following retrotransposition, and as a result have rewired the host regulatory network during the course of evolution. However, it remains unclear whether currently active SINEs contribute to the expansion of TF binding sites. To study the mobility, expression, and function of SINE copies, we first identified about 2,000 insertional polymorphisms of SINE B1 and B2 families within Mus musculus. Using a novel RNA sequencing method developed here, we detected the expression of SINEs in male germ cells at both the subfamily and genomic copy levels: the vast majority of B1 RNAs originated from evolutionarily young subfamilies, whereas B2 RNAs originated from both young and old subfamilies. DNA methylation and chromatin immunoprecipitation-sequencing (ChIP-seq) analyses in liver revealed that polymorphic B2 insertions served as a chromatin boundary element inhibiting the expansion of DNA hypomethylated and histone hyperacetylated regions, and decreased the expression of neighboring genes. Moreover, genomic B2 copies were enriched at the boundary of ChIP-seq peaks of various histone modifications. A total of > 100 polymorphic B2 insertions, together with >10,000 non-polymorphic B2 copies, were bound by CTCF, a well-known chromatin boundary protein. These results suggest that the currently active B2 copies are mobile chromatin boundary elements that can modulate gene expression level, and are likely involved in epigenomic and phenotypic diversification of the mouse species. This novel sequencing method has been designed for expression analysis of medium length (50-300 bp) RNAs, including those of short interspersed elements (SINEs), a class of retrotransposons. After converting the 5' end of transcribed RNAs into a ligatable end, libraries were prepared by a protocol almost same as that for small RNA sequencing. Single-end 300 bp sequencing yielded sequences of the whole length of RNAs.