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SRP067124
Mus musculus
743 Downloadable Samples
Illumina HiSeq 2500, Illumina HiSeq 2000, Illumina HiSeq 1500
Description
Single-cell RNA sequencing (scRNA-seq) offers new possibilities to address biological and medical questions. However, systematic comparisons of the performance of diverse scRNA-seq protocols are lacking. We generated data from 583 mouse embryonic stem cells to evaluate six prominent scRNA-seq methods: CEL-seq2, Drop-seq, MARS-seq, SCRB-seq, Smart-seq and Smart-seq2. While Smart-seq2 detected the most genes per cell and across cells, CEL-seq2, Drop-seq, MARS-seq and SCRB-seq quantified mRNA levels with less amplification noise due to the use of unique molecular identifiers (UMIs). Power simulations at different sequencing depths showed that Drop-seq is more cost-efficient for transcriptome quantification of large numbers of cells, while MARS-seq, SCRB-seq and Smart-seq2 are more efficient when analyzing fewer cells. Our quantitative comparison offers the basis for an informed choice among six prominent scRNA-seq methods and provides a framework for benchmarking further improvements of scRNA-seq protocols. Overall design: J1 mESC in two replicates per library preparation method.
Publication Title
A systematic evaluation of single cell RNA-seq analysis pipelines.
Sample Metadata Fields
Cell line, Subject
View Samples- Homo sapiens
- 81 Downloadable Samples
- Illumina HiSeq 1500
Description
Background Single-cell RNA-sequencing (scRNA-seq) experiments typically analyze hundreds or thousands of cells after amplification of the cDNA. The high throughput is made possible by the early introduction of sample-specific bar codes (BCs), and the amplification bias is alleviated by unique molecular identifiers (UMIs). Thus, the ideal analysis pipeline for scRNA-seq data needs to efficiently tabulate reads according to both BC and UMI. Findings zUMIs is a pipeline that can handle both known and random BCs and also efficiently collapse UMIs, either just for exon mapping reads or for both exon and intron mapping reads. If BC annotation is missing, zUMIs can accurately detect intact cells from the distribution of sequencing reads. Another unique feature of zUMIs is the adaptive downsampling function that facilitates dealing with hugely varying library sizes but also allows the user to evaluate whether the library has been sequenced to saturation. To illustrate the utility of zUMIs, we analyzed a single-nucleus RNA-seq dataset and show that more than 35% of all reads map to introns. Also, we show that these intronic reads are informative about expression levels, significantly increasing the number of detected genes and improving the cluster resolution. Conclusions zUMIs flexibility makes if possible to accommodate data generated with any of the major scRNA-seq protocols that use BCs and UMIs and is the most feature-rich, fast, and user-friendly pipeline to process such scRNA-seq data. Overall design: HEK293T cells were sequenced using the mcSCRB-seq protocol (Bagnoli et al., 2017)
Publication Title
zUMIs - A fast and flexible pipeline to process RNA sequencing data with UMIs.
Sample Metadata Fields
Cell line, Subject
View Samples- Homo sapiens
- 11 Downloadable Samples
- IlluminaHiSeq1500
Description
Many library preparation methods are available for gene expression quantification. Here, we sequenced and analysed Universal Human Reference RNA (UHRR) prepared using Smart-Seq2, TruSeq (public data) and a protocol using unique molecular identifiers (UMIs) that all include the ERCC spike-in mRNAs to investigate the effects of amplification bias on expression quantification. Overall design: UHRR 10 and 12 replicates for Smart-seq2 and UMI-seq library preparation methods, respectively.
Publication Title
The impact of amplification on differential expression analyses by RNA-seq.
Sample Metadata Fields
No sample metadata fields
View Samples- Macaca mulatta, Mus caroli, Mus musculus, Pan troglodytes, Pongo pygmaeus, Homo sapiens, Mus spretus
- 13 Downloadable Samples
Affymetrix Murine Genome U74A Version 2 Array (mgu74av2), Affymetrix Human Genome U95 Version 2 Array (hgu95av2)
Description
Microarray technologies allow the identification of large numbers of expression differences within and between species. Although environmental and physiological stimuli are clearly responsible for changes in the expression levels of many genes, it is not known whether the majority of changes of gene expression fixed during evolution between species and between various tissues within a species are caused by Darwinian selection or by stochastic processes. We find the following: (1) expression differences between species accumulate approximately linearly with time; (2) gene expression variation among individuals within a species correlates positively with expression divergence between species; (3) rates of expression divergence between species do not differ significantly between intact genes and expressed pseudogenes; (4) expression differences between brain regions within a species have accumulated approximately linearly with time since these regions emerged during evolution. These results suggest that the majority of expression differences observed between species are selectively neutral or nearly neutral and likely to be of little or no functional significance. Therefore, the identification of gene expression differences between species fixed by selection should be based on null hypotheses assuming functional neutrality. Furthermore, it may be possible to apply a molecular clock based on expression differences to infer the evolutionary history of tissues.
Publication Title
A neutral model of transcriptome evolution.
Sample Metadata Fields
Sex, Age, Specimen part, Disease, Disease stage
View Samples- Mus musculus
- 17 Downloadable Samples
- Illumina Genome Analyzer IIx
Description
Fbw7, the substrate recognition subunit of SCF(Fbw7) ubiquitin ligase, mediates turnover of multiple proto-oncoproteins and promotes its own degradation. Fbw7-mediated substrate degradation is antagonized by the Usp28 deubiquitinase. We now show, using knockout mice, that Usp28 preferentially deubiquitinates and stabilizes Fbw7. Monoallelic deletion of Usp28 maintains stable Fbw7 but destabilizes Fbw7 substrates. In contrast, complete knockout of Usp28 promotes Pin1-dependent autocatalytic turnover of Fbw7, accumulation of Fbw7 substrates and oncogenic transformation. Overexpression of Usp28 stabilizes both Fbw7 and its substrates and similarly enhances transformation. We propose that dual regulation of Fbw7 activity by Usp28 maintains physiological levels of Fbw7 substrates, and that both loss and overexpression of Usp28 in human cancer promote Fbw7 substrate accumulation. Overall design: RNAseq experiments of E13.5 murine embryonic fibroblasts (MEFs) derived from animals in which Usp28 was either deleted (-/-), wildtype (+/+) or heterozygous (+/-). In a first set of experiments immortalized MEFs of all three genotypes were analysed in biological triplicates. In a second set of experiments immortalized and Ras transformed MEFs of all three genotypes and MEFs which overexpress USP28 (+/+/+) where sequenced in duplicates.
Publication Title
Dual regulation of Fbw7 function and oncogenic transformation by Usp28.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 48 Downloadable Samples
- Affymetrix Murine Genome U74A Version 2 Array (mgu74av2)
Description
Hippocamus and amygdala expression was examined in nave, conditioned stimulus exposed, and fear conditioned mice 30 minutes after behavioral manipulation
Publication Title
Differential transcriptional response to nonassociative and associative components of classical fear conditioning in the amygdala and hippocampus.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 36 Downloadable Samples
- Illumina HiSeq 2500
Description
RNAseq analysis of CD8 T cells becoming dysfunctional in progressing tumors. The overall goal of this study was to elucidate the molecular program that mediates functional unresponsiveness in tumor-specific CD8 T cells. In comparison, we also investigated CD8 T cells differentiating to functional effector and memory T cells during an acute listeria infection. Overall design: T cells were sorted by flow cytometry and RNA-seq was performed.
Publication Title
Chromatin states define tumour-specific T cell dysfunction and reprogramming.
Sample Metadata Fields
Disease, Disease stage, Cell line, Subject
View Samples- Homo sapiens
- 6 Downloadable Samples
- Ion Torrent Proton
Description
Purpose:To identify resistance mechanisms for the chemotherapeutic drug fludarabine in chronic lymphocytic leukemia (CLL), as innate and acquired resistance to fludarabine-based chemotherapy represents a major challenge for long-term disease control. Methods: We employed piggyBac transposon-mediated mutagenesis, combined with next-generation sequencing, to identify genes that confer resistance to fludarabine in a human CLL cell line. Results: RNA-seq profiling of fludarabine-resistant cells suggested deregulated MAPK signaling as involved in mediating drug resistance in CLL. Overall design: To address if the fludarabine-resistant HG3 cells were transcriptionally different at a global level compared to their parental cells, we performed RNA-sequencing of three pairs of HG3 pools
Publication Title
Transposon Mutagenesis Reveals Fludarabine Resistance Mechanisms in Chronic Lymphocytic Leukemia.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 12 Downloadable Samples
- Illumina HiSeq 2000
Description
During development neuronal progenitors compete for growth factors such as nerve growth factor NGF and require the prolyl hydroxylase EglN3 and the kinesin KIF1Bß for developmental apoptosis. Inherited KIF1Bß loss-of-function mutations in neuroblastomas and pheochromocytomas implicate KIF1Bß as a 1p36.2 tumor suppressor, however the mechanism of tumor suppression is unknown. We found that KIF1Bß interacts with the RNA helicase A (DHX9) resulting in DHX9 nuclear accumulation to regulate apoptosis. KIF1Bß-dependent DHX9 nuclear localization leads to transcription of the apoptotic target XIAP-associated factor 1. DHX9 is induced when NGF is limiting and required for apoptosis in cells deprived of NGF. Overall design: NB1 cells were transduced to incorporate shRNA against DHX9 or a scrambled control, and transfected with a KIF1Bß expression vector or control, then transfected cells were isolated and lysed after 48h.
Publication Title
RNA helicase A is a downstream mediator of KIF1Bβ tumor-suppressor function in neuroblastoma.
Sample Metadata Fields
Cell line, Subject
View Samples- Homo sapiens
- 6 Downloadable Samples
- Illumina HiSeq 1500
Description
Purpose: The goal of this study is to investigate the role of CBS enzyme in colorectal carcinogenesis Methods: RNA-Seq transcriptome analysis of CBS-overexpression in NCM356 cels compared to control vector cells Overall design: RNA-seq transcriptome profiling of NCM356-CBS overexpressing cells vs. vector cells
Publication Title
Upregulation of Cystathionine-β-Synthase in Colonic Epithelia Reprograms Metabolism and Promotes Carcinogenesis.
Sample Metadata Fields
Subject
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