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Series GSE171593 Query DataSets for GSE171593
Status Public on Jul 01, 2021
Title Transcriptome-scale analysis for RNAs of medium length, designated as melRNA-seq [RppH-treated]
Organism Mus musculus
Experiment type Non-coding RNA profiling by high throughput sequencing
Summary 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.
 
Overall design 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.
 
Contributor(s) Ichiyanagi K, Mori Y
Citation(s) 34134767
Submission date Apr 06, 2021
Last update date Jul 01, 2021
Contact name Kenji Ichiyanagi
E-mail(s) ichiyana@agr.nagoya-u.ac.jp
Phone +81-52-789-4066
Organization name Nagoya University
Department Graduate School of Bioagricultural Sciences
Lab Laboratory of Genome and Epigenome Dynamics
Street address Furo-cho, Chikusa-ku
City Nagoya
ZIP/Postal code 464-8601
Country Japan
 
Platforms (1)
GPL16417 Illumina MiSeq (Mus musculus)
Samples (2)
GSM5229041 RppH_minus (Intact)
GSM5229042 RppH_plus
Relations
BioProject PRJNA720141
SRA SRP313704

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE171593_RAW.tar 10.0 Kb (http)(custom) TAR (of TXT)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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