Project description:In our study, we generated and sequenced small RNA libraries from commercially available brain total RNA or human blood plasma samples. These samples were generated with MAD-DASH, a method we developed employing CRISPR/Cas9 ribonucleoprotein targeting specific overabundant sequences such as adapter dimer or miRNAs to reduce these sequences from final libraries. We sequenced treated and untreated samples to demonstrate specificity, efficacy, and reproducibility of our MAD-DASH small-RNA sequencing protocol.
Project description:We employed high-throughput sequencing to profile the non-coding RNA content of primary ESC and Fb derived exosomes. All 6 libraries were sequenced to a comparable depth (ESC1: 12.3M, ESC2: 14M, ESC3: 12.8M, FB1: 14.7M, FB2: 13.7M, FB3: 12.3M) and exhibited a very low proportion of adapter-only reads or fragments shorter than 17bp (4.4 ± 0.5% ), showing efficient fragment incorporation during library preparation. All libraries had high small RNA content (>50% successfully aligned and assigned to small RNAs). ESC libraries had significantly higher small RNA content (ESC: 84.5 ± 0.005% vs FB: 60.5 ± 0.05%, p<0.05, two-sided Welch’s t-test). ESC samples show remarkably higher proportions of miRNAs (~9-fold higher, p=3.6*10-5 ) and snRNAs (~12-fold higher, p=0.002) compared to Fb. Fibroblasts exhibited higher RPM of reads mapping on lncRNAs (4-fold, p=0.052)
Project description:MicroRNA (miRNA) and other types of small regulatory RNAs play a crucial role in the regulation of gene expression in eukaryotes. Several distinct classes of small regulatory RNAs have been discovered in recent years. To extend the repertoire of small regulatory RNAs characterized in chickens we used a deep sequencing approach developed by Solexa (now Illumina Inc.). We sequenced three small RNA libraries prepared from different developmental stages of the chicken embryo (days 5, 7, and 9) to produce over 9.5 million short sequence reads. We developed a bioinformatics pipeline to distinguish authentic mature miRNA sequences from other classes of small RNAs and short RNA fragments represented in the sequencing data. Using this approach we detected almost all of the previously known chicken miRNAs and their respective miRNA* sequences. In addition we discovered 449 putative new chicken miRNAs. Of these, 430 miRNAs appear to be specific to the avian lineage. Another 6 new miRNAs had evidence of evolutionary conservation in at least one vertebrate species outside of the bird lineage. The remaining 13 putative miRNAs appear to represent chicken orthologs of known vertebrate miRNAs. We discovered 39 additional putative miRNA candidates originating from miRNA generating intronic sequences known as mirtrons. Keywords: miRNA discovery, mirtrons, chicken embryo
Project description:Collection of various small RNA libraries of C. Reinhardtii sequenced using Illumina sRNA sequencing. Libraries are derived from various C. Reinhardtii genotypes (CC1883, CC4350, CC125, J), ecotypes, life cycles (Vegatative, Zygote) treatments and mutants. Preparation was done over a period of several years as a continuous effort to build up a comprehensive collection.
Project description:Many evolutionarily conserved miRNAs in plants regulate transcription factors with key functions in plant development. Hence, mutations in core components of the miRNA biogenesis machinery causes strong growth defects. An essential aspect of miRNA biogenesis is the precise excision of the small RNA from an arm of the fold-back precursor. Defects in the selection of the correct miRNA sequence will ultimately affect miRNA target specificity, plant development and other processes controlled by these small RNAs. Intriguingly, plant miRNA precursors are largely variable in size and shape and can be processed by different modes. Here, we optimized genomic approaches to detect processing intermediates during miRNA biogenesis. We identified and characterized an endogenous miRNA whose processing is triggered by a terminal branched loop. Plant miRNA processing can be initiated by internal bubbles, small terminal loops or branched loops followed by dsRNA segments of 15-17 bp. Interestingly, precision and efficiency vary with the processing modes suggesting intrinsic differences between miRNA biogenesis pathways. Despite the various potential structural determinants present in a single a miRNA precursor, we found that DCL1 is mostly guided by a predominant structural region in each precursor in wild-type plants. However, genomic studies of miRNA processing intermediates in fiery1, hyl1 and se mutants revealed the existence of cleavage signatures consistent with the recognition of alternative or cryptic processing determinants in miRNA precursors. The results provide a general view of the mechanisms underlying the specificity of miRNA biogenesis in plants.
Project description:High-throughput sequencing of Drosophila melanogaster small RNAs from OSS cells. total RNA, ~18-26nt RNAs isolated using PAGE, ligation to adapters requires 5' monophosphate and 3' OH For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf