Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

ABSTRACT: We report a novel single-cell total RNA-seq method, RamDA-seq, by combining a novel reverse transcription (RT) technology, RT with random displacement amplification (RT-RamDA), and not-so-random (NSR) primers. RT-RamDA provides global cDNA amplification directly from RNA during RT without any universal adopters, which benefits RT efficiency, simplification of the procedure, avoiding the step of PCR amplification, and decontamination of genomic DNA. NSR enables random priming while preventing cDNA synthesis from rRNAs. RamDA-seq showed high sensitivity to non-poly(A) RNAs and full-length coverage even for extremely long transcripts (>10 kb). Moreover, RamDA-seq revealed recursive splicing, a multi-step, splicing, within > 300 kbp-long introns. Finally, RamDA-seq enabled the first genome-wide analysis of enhancer RNAs (eRNAs) in single cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE98664 | GEO | 2018/02/12

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

A novel global cDNA amplification technology directly from an RNA template via reverse transcription

Project description:We developed a method, Reverse Transcription with Random Displacement Amplification (RT-RamDA), to reverse transcribe and amplify cDNA directly from RNA templates using strand displacement and RNA-dependent DNA polymerase activity. This method resulted in a cDNA yield approximately 100-fold higher than conventional RT with high sensitivity, enabling gene expression profiling of low-expression genes with a small amount of RNA, such as that obtained at the single-cell level.

2019-01-01 | GSE108463 | GEO

Dam-specific RNA-seq

Project description:To determine the transcription start site and the abundance of dam mRNA, we conducted mRNA-specific RNA-Seq experiments. Essentially two strategies were tested. First strategy: We obtained the cDNA with a specific RT-PCR in order to get the mRNA from our dam construct (see DNA-seq of DamID reporter cassette). An enrichment, in which we used a Biotinylated dam oligo and Streptavidin magnetic beads, was needed. Then, similar to SHAPE-seq, the ssDNA ligation was performed with CircLigase. The last step was the PCR reaction in order to prepare the cDNA libraries to be sequenced. Second strategy: Similar to the first one, but the specificity was introduced at the PCR, not at the RT. Thereby, busk cDNA was prepared with random hexamers. The enrichment step was not needed.

2018-03-22 | E-MTAB-5363 | biostudies-arrayexpress

Identification of stably expressed reference genes for expression studies in Arabidopsis thaliana using mass spectrometry-based label-free quantification

Project description:Arabidopsis thaliana has been used regularly as a model plant in gene expression studies on transcriptional reprogramming upon pathogen infection, such as that by Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), or when subjected to stress hormone treatments including jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). RT-qPCR has been extensively employed to quantitate these gene expression changes. However, the accuracy of the quantitation is largely dependent on the stability of the expressions of reference genes used for normalization. Recently, RNA-seq has been widely used to mine stably expressed genes for use as references in RT-qPCR. However, the amplification step in RNA-seq creates an intrinsic bias against those genes with relatively low expression levels, and therefore does not provide an accurate quantification of all expressed genes. In this study, we employed mass spectrometry-based label-free quantification (LFQ) in proteomic analyses to identify those proteins with abundances unaffected by Pst DC3000 infection. We verified, using RT-qPCR, that the levels of their corresponding mRNAs were also unaffected by Pst DC3000 infection. In addition, using RT-qPCR, we verified that the mRNAs were stably expressed upon stress hormone treatments including JA, SA, and ABA. Results indicated that the candidate genes identified here had stable expressions upon these stresses and are suitable to be used as reference genes for RT-qPCR. Among the 18 candidate reference genes reported in this study, many of them had greater expression stability than the commonly used reference genes, such as ACT7, in previous studies. Here, besides proposing more appropriate reference genes for Arabidopsis expression studies, we also demonstrated the capacity of mass spectrometry-based LFQ to quantify protein abundance and the possibility to extend protein expression studies to the transcript level.

2022-09-05 | PXD035677 | Pride

Integrating whole transcriptome assays on a lab-on-a-chip for single cell gene profiling

Project description:To correlate gene expression profiles to fundamental biological processes such as cell growth, differentiation and migration, it is essential to work at the single cell level. Gene expression analysis always starts with the relatively low efficient reverse transcription (RT) of RNA into complementary DNA (cDNA), an essential step as unprocessed RNAs will not be analyzed further. In this paper, we present a novel method for RT that uses microfluidics to manipulate nanoliter volumes. We compare our method to conventional protocols performed in microliter volumes. More specifically, reverse transcription was performed either in a poly-dimethylsiloxane (PDMS) rotary mixer or in a tube, using single cell amount of mouse brain RNA (10 pg), and was followed by a template-switching PCR (TS-PCR) amplification step. We demonstrate that, using the microfluidic protocol, 74% of the genes expressed in mouse brain were detected, while only 4% were found with the conventional approach. We next profiled single neuronal progenitors. Using our microfluidic approach, i.e. performing cell capture, lysis and reverse transcription on-chip followed by TS-PCR amplification in tube, a mean of 5000 genes were detected in each neuron, which corresponds to the expected number of genes expressed in a single cell. This demonstrates the outstanding sensitivity of the microfluidic method. Keywords: Single cell, transcriptome, PDMS, microfluidic, reverse-transcription (RT), template-switching, lab-on-a-chip

2008-02-15 | GSE6941 | GEO

Digital RNA Sequencing Minimizes Sequence-Dependent Bias and Amplification Noise with Optimized Single Molecule Barcodes

Project description:RNA-Seq is a powerful tool for transcriptome profiling, but is hampered by sequence-dependent bias and inaccuracy at low copy numbers intrinsic to exponential PCR amplification. We developed a simple strategy for mitigating these complications, allowing truly digital RNA-Seq. Following reverse transcription, a large set of barcode sequences is added in excess, and nearly every cDNA molecule is uniquely labeled by random attachment of barcode sequences to both ends. After PCR, we applied paired-end deep sequencing to read the two barcodes and cDNA sequences. Rather than counting the number of reads, RNA abundance is measured based on the number of unique barcode sequences observed for a given cDNA sequence. We optimized the barcodes to be unambiguously identifiable even in the presence of multiple sequencing errors. This method allows counting with single copy resolution despite sequence-dependent bias and PCR amplification noise, and is analogous to digital PCR but amendable to quantifying a whole transcriptome. We demonstrated transcriptome profiling of E. coli with more accurate and reproducible quantification than conventional RNA-Seq.

2012-01-07 | GSE34449 | GEO

Single-cell RNA Sequencing of Murine Lungs to Compare Two Different Models of Alveolar Simplification

Project description:Neonatal hyperoxia exposure causes alveolar simplification in mice and has been employed as a model to study bronchopulomonary dysplasia, or chronic lung disease of prematurity. Loss of epithelial TGF-beta signaling has also been shown to cause alveolar simplification in mouse models of lung development. We used single cell RNA sequencing (scRNA-seq) to analyze both of these models of lung injury to identify shared molecular and cellular mechanisms that contribute to pathogenic alveolar simplification.

2024-03-14 | GSE243129 | GEO

Analysis of single cell gene expression profile in human iPSC-derived lt-NES cells

Project description:We performed single-cell total RNA-seq of iPSC-derived neuroepitherial-like stem cells using RamDA-seq methods. We observed hetelogeneity and classified it based on differentiation potential.

2019-10-23 | GSE136611 | GEO

TP53 and TP63 modulation of Squamous PDAC

Project description:Based on the association of TP53 mutation and upregulated TP63 expression in the squamous subtype, we used cell lines derived from genetically engineered mouse models of PDAC (KRAS Trp53fl/+ and KRAS Trp53fl/+ Trp63fl/fl) to begin to unravel the functional consequences of these events in defining squamous tumours. RNA-Seq libraries were generated using TruSeq Stranded Total RNA (Part no. 15031048 Rev. D April 2013) kits, using on a Perkin ElmeraTMs Sciclone G3 NGS Workstation (Product no. SG3-31020-0300). Ribosomal depletion step was performed on 1 ug of total RNA using Ribo-Zero Gold prior to a heat fragmentation step aimed at producing libraries with an insert size between 120-200 bp. cDNA was then synthesized from the enriched and fragmented RNA using InvitrogenaTMs SuperScript II Reverse Transcriptase (Catalog no. 18064) and random primers. The resulting cDNA was further converted into double stranded DNA in the presence of dUTP to prevent subsequent amplification of the second strand and thus maintain the strandedness of the library. Following 3aTM adenylation and adaptor ligation libraries were subjected to 15 cycles of PCR to produce RNA-Seq libraries ready for sequencing. Prior to sequencing, exome and RNA-Seq libraries were qualified and quantified via CaliperaTMs LabChip GX (Part no. 122000) instrument using the DNA High Sensitivity Reagent kit (Product no. CLS760672). Quantification of libraries for clustering was performed using the KAPA Library Quantification Kits For Illumina sequencing platforms (Kit code KK4824) in combination with Life Technologies Viia 7 real time PCR instrument. All libraries were sequenced using the Illumina HiSeq 2000/2500 system with TruSeq SBS Kit v3 - HS.

2016-03-03 | E-MTAB-4415 | biostudies-arrayexpress

Digital RNA Sequencing Minimizes Sequence-Dependent Bias and Amplification Noise with Optimized Single Molecule Barcodes

2012-01-07 | E-GEOD-34449 | biostudies-arrayexpress

Transcriptome sequencing Analysis of hLFSCs with and without mechanical stress

Project description:Purpose:The goals of this study are to MS (mechanical stress) how to change the cellular pathway of hLFSCs (human ligament flavum stem cells). Methods: Extract hLFSCs from the ligament flavum of patients.Treat the cells with and without (in control)MS.Total RNA was extracted from cells with Tripure Isolation Reagent.Subsequently, RNA was purified through rRNA depletion and then subjected to cDNA synthesis and RNA amplification. Next, random hexamer primer cDNA libraries were evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and sequenced on Illumina Hiseq 4000 sequencing platform (Illumina, San Diego, California, USA) following the manufacturer’s instructions for paired-end 150 bp reads (Lifegenes, Shanghai, China).

2021-10-13 | GSE185614 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data