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Next Generation Sequencing Facilitates Quantitative Analysis of the lncRNA Transcriptome in 6 Distinct Germ Cell Types During Mouse Spermatogenesis

ABSTRACT: Purpose: The goals of this study are to obtain the lncRNA landscape in 6 distinct germ cell types during mouse spermatogenesis. Methods: RNA-seq data of lncRNAs from 6 distinct cell types were generated by deep sequencing using Illumina HiSeq 2500. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks. qRT-PCR validation was performed using SYBR Green assays. Results: Using the rRNA minus strategy, we generated a total of 1100.65 Gb paired and sequence reads with a length of 150 bp. Next, we trimmed the adaptor sequence and filtered the sequence data for low-quality reads at a high stringency with an average Phred quality score less than 20, which resulted in a total of 745.67 Gb high-quality sequence reads. After filtering the reads, almost all sequence reads had a Phred quality score higher than 20, and the percentage of Ns in the reads was nearly zero. By using unique whole-genome alignment (mouse genome (mm10)), a total of 14587 mRNA genes and 6921 lncRNA genes were expressed as fragments per kilobase of exon per million reads mapped (FPKM)>0.1 in at least one sample. In addition, to obtain fingerprint lncRNAs during spermatogenesis, we first identified 2327 differentially expressed lncRNAs between every two adjacent stages. Furthermore, we identified 437 fingerprint lncRNAs that were expressed at a FPKM > 10 of its highly expressed stage. Conclusions: Our RNA-seq dataset comprehensively dissects the dynamic expression mode of lncRNAs during mouse spermatogenesis and generated 28 relatively robust potential lncRNA candidates in spermatogenesis.

ORGANISM(S): Mus musculus

PROVIDER: GSE145130 | GEO | 2020/11/16

REPOSITORIES: GEO

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Project description:Only a minuscule fraction of long non-coding RNAs (lncRNAs) are well characterized. The evolutionary history of lncRNAs can provide insights into their functionality, but comparative analyses have been precluded by our ignorance of lncRNAs in non-model organisms. Here, we use RNA sequencing to identify lncRNAs in eleven tetrapod species and we present the first large-scale evolutionary study of lncRNA repertoires and expression patterns. We identify ~11,000 primate- specific lncRNA families, which show evidence for selective constraint during recent evolution, and ~2,400 highly conserved lncRNAs (including ~400 genes that likely originated more than 300 million years ago). We find that lncRNAs, in particular ancient ones, are generally actively regulated and may predominantly function in embryonic development. lncRNA X-inactivation patterns reveal an extremely female-biased monotreme-specific lncRNA, which may partially compensate X-dosage in this lineage. Most lncRNAs evolve rapidly in terms of sequence and expression levels, but global patterns like tissue specificities are often conserved. We compared expression patterns of homologous lncRNA and protein-coding families across tetrapods to reconstruct an evolutionarily conserved co-expression network. This network, which surprisingly contains many lncRNA hubs, suggests potential functions for lncRNAs in fundamental processes like spermatogenesis or synaptic transmission, but also in more specific mechanisms such as placenta growth suppression through miRNA production. [Batch 1 and 2] To broaden our understanding of lncRNA evolution, we used an extensive RNA-seq dataset to establish lncRNA repertoires and homologous gene families in 11 tetrapod species. We analyzed the poly- adenylated transcriptomes of 8 organs (cortex/whole brain without cerebellum, cerebellum, heart, kidney, liver, placenta, ovary and testis) and 11 species (human, chimpanzee, bonobo, gorilla, orangutan, macaque, mouse, opossum, platypus, chicken and the frog Xenopus tropicalis), which shared a common ancestor ~370 millions of years (MY) ago. Our dataset included 47 strand-specific samples, which allowed us to confirm the orientation of gene predictions and to address the evolution of sense-antisense transcripts. See also GSE43721 (Soumillon et al, Cell Reports, 2013) for three strand-specific samples for mouse brain, liver and testis.

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Next Generation Sequencing Facilitates Quantitative Analysis of the lncRNA Transcriptome in 6 Distinct Germ Cell Types During Mouse Spermatogenesis

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