Transcriptome-scale analysis for RNAs of medium length, designated as melRNA-seq [RppH-treated]

Small interspersed elements (SINEs) is transcribed by RNA polymerase III (Pol III) to produce RNAs of typically 100 to 500 nucleotides in length. Although the abundance of SINE RNAs can be analyzed by Northern blotting and primer extension, the nature (sequence, exact length, and genomic origin) of these RNAs cannot be revealed by these methods. Moreover, mRNA sequencing (mRNA-seq) is not able to distinguish bona fide SINE RNAs and SINE sequences present in longer transcripts. To elucidate the abundance, source loci, and sequence nature of SINE RNAs, we have established a deep sequencing method, designated as melRNA-seq (medium length RNA-seq), which can determine whole-length sequences of RNAs. The total RNA samples were treated with 5' Pyrophosphohydrolase (RppH), which allowed ligation of an RNA adaptor to the 5’ end of intact SINE RNAs. Another adaptor was ligated to the 3’ end, followed by reverse transcription, PCR amplification, size selection, and single-end deep sequencing. Analysis of two biological replicates of RNAs from mouse spermatogonia showed high reproducibility of the SINE expression data both at family level and locus level. Therefore, this new method can be used for the quantification and detailed sequence analysis of medium length non-coding RNAs, such as rRNA, snRNA, tRNAs, and SINE RNAs. The dynamic range is much wider than Northern blotting and primer extension. 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.