Single cell RNA-seq data of human hESCs comparing experiments with cDNA library equalization
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PROVIDER: PRJNA658114 | ENA |
REPOSITORIES: ENA
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Project description:Considerable effort has been devoted to refining experimental protocols having reduced levels of technical variability and artifacts in single-cell RNA-sequencing data (scRNA-seq). We here present evidence that equalizing the concentration of cDNA libraries prior to pooling, a step not consistently performed in single-cell experiments, improves gene detection rates, enhances biological signals, and reduces technical artifacts in scRNA-seq data. In this submission the original single-cell cDNA is from GSE75748 cells labelled EC2 and TB2 in which the cDNA was not equalized prior to library preparation. We performed a set of experiments with the equalization step and provide three count matrices - 1) EQ (equalized cDNA prior to pooling in equal amounts); 2) EQ Vary (same as EQ but pooled at unequal amounts); 3) EQ-75% (equalized cDNA and pooled equally, sequeced to lower total depth).
2021-11-03 | GSE156494 | GEO
Project description:cDNA microarrays have become the technology of choice for profiling the expression of thousands of genes simultaneously in a single sample. However, cross-hybridization between targets and probes that do not correspond must be considered carefully during the design, analysis, and interpretation of these experiments. Here, we examined this potentially confounding issue by competitively hybridizing fragments with similar sequence obtained from two paralogous genes to a custom cDNA microarray that contains probes for only one of these two genes. We found that although a large contiguous portion of these two fragments was 84% identical and only one target was represented on the array, there was very little cross-hybridization of the non-represented target to either the paralogous probes or other (non-similar) features. We conclude that cDNA microarrays are indeed very specific and that promiscuous paralog hybridization adds little noise to the data. More generally, the absence of specific probes for any given gene does not increase the chance for cross-hybridization between non-specific probes and targets, even if sequence similarity is relatively high.
2009-07-03 | GSE16939 | GEO
Project description:While a first draft of the equine genome is available and predictions are made regarding resulting genes and proteins, little is known about the actual transcriptome. So far, published expressed sequence tags (ESTs) from different horse tissues were generally rather short (≤600bp) and hardly annotated, reflecting the problem that good cDNA libraries are very difficult to analyse. In this approach, we aimed to establish and analyse a normalised immune cell cDNA library (using freshly isolated and activated lymphocytes, NK cells, monocytes and DC). In particular, we wanted to test next generation sequencing combined with a series of bioinformatic approaches. The resulting cDNA library contained 2x107 clones of which 1056 were used for an initial Sanger sequencing and 4x106 for the deep sequencing analysis. Through the latter we obtained >29k sequences for which more than 5000 matches where found on the equine reference sequences. Additionally we could identify more than 3500 sequences which had matches on both - non-equine RNA sequences as well as the equine genome. In these we find both extensions of existing RefSeq models and novel mRNAs alike. Less than 2% of sequences did not have any match in the mentioned databases.
2012-06-30 | GSE26794 | GEO
Project description:We constructed retroviral cDNA library of 176 proto-oncogenes and HOX genes to screen for the leukemia initiating gene combinations. The present submission is the NGS data of the PCR products of the whole inserts from the library itself and the bone marrow cells infected with retroviral library
2016-11-10 | GSE74806 | GEO
Project description:We deep sequenced a degradome library constructed from different soybean tissues. As a result, 19,830,257 represented 5,337,590 distinct signatures were obtained. 70.98% of the signatures were assigned to one soybean cDNA sequence and 24.05% matched with two cDNA sequences. 428 potential targets of small RNAs and 25 novel miRNA families were identified in soybean. A total of 211 potential miRNA targets including 150 conserved miRNA targets and 69 soybean-specific miRNA targets were identified. The signatures distribution on soybean primary miRNAs (pri-miRNAs) showed that most of the pri-miRNAs had the characteristic pattern of Dicer processing. The TAS3 small RNAs (siRNAs) biogenesis was conserved in soybean and nine Auxin Response Factors (ARFs) were identified as the TAS3 siRNA targets. The global identification of miRNAs targets would contribute to the functional research of the miRNA in soybean.
2011-11-02 | GSE33379 | GEO
Project description:Deep sequencing of small RNAs and degradome cDNA library in soybean
| PRJNA149105 | ENA
Project description:We deep sequenced a degradome library constructed from different soybean tissues. As a result, 19,830,257 represented 5,337,590 distinct signatures were obtained. 70.98% of the signatures were assigned to one soybean cDNA sequence and 24.05% matched with two cDNA sequences. 428 potential targets of small RNAs and 25 novel miRNA families were identified in soybean. A total of 211 potential miRNA targets including 150 conserved miRNA targets and 69 soybean-specific miRNA targets were identified. The signatures distribution on soybean primary miRNAs (pri-miRNAs) showed that most of the pri-miRNAs had the characteristic pattern of Dicer processing. The TAS3 small RNAs (siRNAs) biogenesis was conserved in soybean and nine Auxin Response Factors (ARFs) were identified as the TAS3 siRNA targets. The global identification of miRNAs targets would contribute to the functional research of the miRNA in soybean. one sample, We deep sequenced a degradome library constructed from different soybean tissues.
2011-11-02 | E-GEOD-33379 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE33378: Deep sequencing of small RNAs from different tissues in soybean GSE33379: Deep sequencing of the degradome cDNA library in soybean Refer to individual Series
2011-11-02 | E-GEOD-33380 | biostudies-arrayexpress
Project description:While a first draft of the equine genome is available and predictions are made regarding resulting genes and proteins, little is known about the actual transcriptome. So far, published expressed sequence tags (ESTs) from different horse tissues were generally rather short (?600bp) and hardly annotated, reflecting the problem that good cDNA libraries are very difficult to analyse. In this approach, we aimed to establish and analyse a normalised immune cell cDNA library (using freshly isolated and activated lymphocytes, NK cells, monocytes and DC). In particular, we wanted to test next generation sequencing combined with a series of bioinformatic approaches. The resulting cDNA library contained 2x107 clones of which 1056 were used for an initial Sanger sequencing and 4x106 for the deep sequencing analysis. Through the latter we obtained >29k sequences for which more than 5000 matches where found on the equine reference sequences. Additionally we could identify more than 3500 sequences which had matches on both - non-equine RNA sequences as well as the equine genome. In these we find both extensions of existing RefSeq models and novel mRNAs alike. Less than 2% of sequences did not have any match in the mentioned databases. 1 pooled set of samples from one animal analysed
2012-06-29 | E-GEOD-26794 | biostudies-arrayexpress
Project description:Here, we used high-resolution mass spectrometry to identify differentially expressed RNA-binding proteins (RBPs) between TE03 (I3) human embryonic stem cells (hESCs) and human foreskin fibroblasts (HFFs). 2102Ep embryonal carcinoma (EC) cells were used as a reference for pluripotent cells. This data accompanies the manuscript: "Uncovering the RNA-binding protein landscape in the pluripotency network of human embryonic stem cells". Abstract: "Embryonic stem cell (ESC) self-renewal and cell-fate decisions are driven by a broad array of molecular signals. While transcriptional regulators have been extensively studied in human ESCs (hESCs), the extent to which RNA-binding proteins (RBPs) contribute to human pluripotency remains unclear. Here, we carry out a proteome-wide screen and identify 810 proteins that directly bind RNA in hESCs. We reveal that RBPs are preferentially expressed in hESCs and dynamically regulated during exit from pluripotency and early lineage specification. Moreover, we show that nearly 200 RBPs are affected by knockdown of OCT4, a master regulator of pluripotency, several dozen of which are directly bound by this factor. Intriguingly, over 20 percent of the proteins detected in our study are putative DNA- and RNA-binding proteins (DRBPs), among them key transcription factors (TFs). Using fluorescently labeled RNA and seCLIP (single-end enhanced crosslinking and immunoprecipitation) experiments, we discover that the pluripotency-associated STAT3 and OCT4 TFs interact with RNA in hESCs and confirm the direct binding of STAT3 to the conserved NORAD long-noncoding RNA. Taken together, our findings indicate that RBPs have a more widespread role in human pluripotency than previously appreciated, reinforcing the importance of post-transcriptional regulation in stem cell biology".
2023-05-05 | PXD025284 | Pride