A unified strategy to clone modified/unmodified small RNAs and mRNAs for high throughput sequencing
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ABSTRACT:
ORGANISM(S): Caenorhabditis elegans
PROVIDER: GSE129664 | GEO | 2020/01/24
REPOSITORIES: GEO
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2013-04-22 | E-GEOD-44399 | biostudies-arrayexpress
Project description:Protein translation is at the heart of cellular metabolism and its in-depth characterization is key for many lines of research. Recently, ribosome profiling became the state-of-the-art method to quantitatively characterize translation dynamics at a transcriptome-wide level. However, the strategy of library generation affects its outcomes. Here, we present a modified ribosomeprofiling protocol starting from yeast, human cells and vertebrate brain tissue. We use a DNA linker carrying four randomized positions at its 5â and a reverse-transcription (RT) primer with three randomized positions to reduce artifacts during library preparation. The use of seven randomized nucleotides allows to efficiently detect library-generation artifacts. We find that the effect of polymerase chain reaction (PCR) artifacts is relatively small for global analyses when sufficient input material is used. However, when input material is limiting, our strategy improves the sensitivity of gene-specific analyses. Furthermore, randomized nucleotides alleviate the skewed frequency of specific sequences at the 3â end of ribosome-protected fragments (RPFs) likely resulting from ligase specificity. Finally, strategies that rely on dual ligation show a high degree of gene-coverage variation. Taken together, our approach helps to remedy two of the main problems associated with ribosome-profiling data. This will facilitate the analysis of translational dynamics and increase our understanding of the influence of RNA modifications on translation. Ribosome profiling and mRNA-seq libraries from wt yeast comparing different library preparation approaches using different combinations of randomized and non-randomized linkers and RT primers.
2016-08-01 | E-GEOD-84746 | biostudies-arrayexpress
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2019-08-21 | PXD014066 | Pride
Project description:High-throughput sequencing of Drosophila pseudoobscura and Drosophila simulans small RNAs. ~18-26nt RNAs were isolated from total RNA using PAGE, ligation to adapters requires 5' monophosphate and 3' OH. Small RNAs were cloned from Drosophila pseudoobscura (heads and pooled 0-12 and 12-24 hour embryos) and Drosophila simulans (pooled 0-12 and 12-24 hour embryos). Sequencing was performed using the Illumina 1G platform. Following removal of 3' linker sequences, the clipped sequences longer than 18 nt were kept.
2009-11-24 | E-GEOD-13677 | biostudies-arrayexpress
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2022-08-15 | PXD031150 | Pride
Project description:Small RNAs play crucial roles in regulation of gene expression in many eukaryotes. Here, we report the cloning and characterization of 18-26 nt RNAs in the social amoeba Dictyostelium discoideum. This survey uncovered developmentally regulated microRNA candidates whose biogenesis, at least in one case, is dependent on a Dicer homolog, DrnB. Furthermore, we identified a large number of 21 nt RNAs originating from the DIRS-1 retrotransposon, clusters of which have been suggested to constitute centromeres. Small RNAs from another retrotransposon, Skipper, were significantly up-regulated in strains depleted of the second Dicer-like protein, DrnA, and a putative RNA-dependent RNA polymerase, RrpC. In contrast, the expression of DIRS-1 small RNAs was not altered in any of the analyzed strains. This suggests the presence of multiple RNAi pathways in D. discoideum. In addition, we isolated several small RNAs with antisense complementarity to mRNAs. Three of these mRNAs are developmentally regulated. Interestingly, all three corresponding genes express longer antisense RNAs from which the small RNAs may originate. In at least one case, the longer antisense RNA is complementary to the spliced but not the unspliced pre-mRNA, indicating synthesis by an RNA-dependent RNA polymerase. Keywords: cDNA library; small RNA sequencing The aim of this study was to investigate the small RNA (18-26 nt) profile of Dictyostelium discoideum during growth and development. For this reason, we cloned and sequenced pooled small RNAs from growing single cells and from two different multicellular stages (16 and 24 hours of development). cDNA libraries of 18-26 nt D. discoideum RNAs were constructed according to two different protocols (Lee, R.C. and Ambros, V. (2001) Science, 294; Lau, N.C. et al (2001) Science, 294). Briefly, total RNA was isolated from growing D. discoideum AX4 strain cells as well as from cells developed for 16 hours and 24 hours, and the fractions were subsequently pooled. After size fractionation and ligation of a 3’ linker, the RNA was divided into two fractions, one of which was directly ligated to a 5’ linker, thus selecting for small RNAs with 5’ monophosphates. Following RT-PCR, the PCR fragments were cloned and sequenced. The second fraction was subjected to reverse transcription directly after 3’ ligation. Apart from synthesizing a complementary DNA strand, the reverse transcriptase adds a few non-templated C residues at the 3’ end. These C:s were then hybridized to a DNA oligo with three 3’ G:s followed by RT-PCR, cloning and sequencing. This approach is insensitive of the nature of the 5’ end of the small RNA.
2007-10-16 | E-GEOD-8755 | biostudies-arrayexpress
Project description:Endogenous 24nt short interfering RNAs (siRNAs) derived mostly from intergenic and repetitive genomic regions constitute a major class of endogenous small RNAs in Arabidopsis thaliana. Accumulation of A. thaliana 24nt siRNAs requires the Dicer family member DCL3, and clear homologs of DCL3 exist in both flowering and non-flowering plants. However, the absence of a conspicuous 24nt peak in the total RNA populations of several non-flowering plants has raised the question of whether this class of siRNAs might, in contrast to the ancient 21nt microRNAs (miRNAs) and 21-22nt trans-acting siRNAs (tasiRNAs), be an angiosperm-specific innovation. Analysis of non-miRNA, non-tasiRNA hotspots of small RNA production within the genome of the moss Physcomitrella patens revealed multiple loci which consistently produced a mixture of 21-24nt siRNAs with a peak at 23nts. These Pp23SR loci were significantly enriched in transposon content, depleted in overlap with annotated genes, and typified by dense concentrations of the 5-methyl cytosine (5mC) DNA modification. Deep sequencing of small RNAs from two independent Ppdcl3 mutants showed that the P. patens DCL3 homolog is required for the accumulation of 22-24nt siRNAs, but not 21nt siRNAs, at Pp23SR loci. The 21nt component of Pp23SR-derived siRNAs was also unaffected by a mutation in the RNA-dependent RNA polymerase mutant Pprdr6. Transcriptome-wide, Ppdcl3 mutants specifically failed to accumulate 23 and 24nt small RNAs from repetitive regions. We conclude that intergenic/repeat-derived siRNAs are indeed a broadly conserved, distinct class of small regulatory RNAs within land plants. Our results also suggest that Pp DCL3 produces siRNAs of heterogenous size, unlike its A. thaliana homolog which generates exclusively 24nt siRNAs. Small RNAs from Wild-type (two technical replicates), rdr6-19 (two technical replicates), dcl3-5 (one sample), and dcl3-10 (one sample) were sequenced using a Solexa GA. Three different linkers were used for different samples and mixed prior to sequencing. linker 1: CTGTAGGCACCATCAAT (GPL7174 AND GSM313212, GSM313213) linker 2: CACTCGGGCACCAAGGA (GPL7175 AND GSM313214, GSM313215) linker 3: TTTAACCGCGAATTCCAG (GPL7176 AND GSM313216, GSM313217) Raw data: Two FASTQ files represent the raw data underlying the processed sample data in GSE12468. 080616_s_7_seq_GAK-2.fastq corresponds to GSM313212, GSM313214, and GSM313216. 080616_s_8_seq_GAK-3.fastq corresponds to GSM313213, GSM313215, and GSM313217. Experimentally, samples GSM313212, GSM313214, and GSM313216 (i.e., all three genotypes) were sequenced in the same run (hence the same FASTQ file). The sequencing run was a mixture of three different libraries with different linker sequences (as indicated above). These 'barcoded' samples were computationally extracted based on the linker sequences. This is also true for samples GSM313213, GSM313215, and GSM313217. Raw data were filtered to identify small RNAs associated with each linker. Subsequently, small RNAs were filtered to remove rRNA fragments. The remainder were mapped to the Physcomitrella patens draft genome version 1.2 (using oligomap) and filtered to retain only those with one or more perfect match. See Cho et al. for details.
2008-12-19 | E-GEOD-12468 | biostudies-arrayexpress
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2010-05-27 | E-GEOD-20575 | biostudies-arrayexpress
Project description:Intervention type:DRUG. Intervention1:Huaier, Dose form:GRANULES, Route of administration:ORAL, intended dose regimen:20 to 60/day by either bulk or split for 3 months to extended term if necessary. Control intervention1:None.
Primary outcome(s): For mRNA libraries, focus on mRNA studies. Data analysis includes sequencing data processing and basic sequencing data quality control, prediction of new transcripts, differential expression analysis of genes. Gene Ontology (GO) and the KEGG pathway database are used for annotation and enrichment analysis of up-regulated genes and down-regulated genes.
For small RNA libraries, data analysis includes sequencing data process and sequencing data process QC, small RNA distribution across the genome, rRNA, tRNA, alignment with snRNA and snoRNA, construction of known miRNA expression pattern, prediction New miRNA and Study of their secondary structure Based on the expression pattern of miRNA, we perform not only GO / KEGG annotation and enrichment, but also different expression analysis.. Timepoint:RNA sequencing of 240 blood samples of 80 cases and its analysis, scheduled from June 30, 2022..
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