Browse
Submit Data
Databases
API
Help

Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

15 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Evaluating bias-reducing protocols for RNA sequencing library preparation

ABSTRACT: The ligation step in RNA sequencing library generation is a known source of bias. We present the first comparison of the standard duplex adaptor protocol supplied by Life Technologies for use on the Ion Torrent PGM with an alternate single adaptor approach involving CircLigase (CircLig). We also investigate whether using the thermostable ligase Methanobacterium thermoautotrophicum RNA ligase K97A (Mth K97A) for the initial ligation step in the CircLigase protocol reduces bias. A pool of small RNA fragments of known composition was converted into a sequencing library using one of three protocols and sequenced on an Ion Torrent PGM. The single adaptor CircLigase-based approach significantly reduces, but does not eliminate, bias in Ion Torrent data. Using Mth K97A as part of the CircLig method does not further reduce bias.

INSTRUMENT(S): Ion Torrent PGM

ORGANISM(S): synthetic construct

SUBMITTER: Ruth Spriggs

PROVIDER: E-MTAB-2566 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Json Xml

Similar Datasets

Using GRO-seq experiments to determine the mechanism of C. elegans dosage compensation's control of transcription

Project description:Dosage Compensation is required to correct for uneven gene dose between the sexes. We utilized global run-on sequencing (GRO-seq) to examine how Caenorhabditis elegans dosage compensation reduces transcription of X-linked genes. To facilitate these experiments, we required accurate 5M-bM-^@M-^Y-ends of genes that have been missing due to a co-transcriptional trans-splicing event common in nematodes. We developed a modified GRO-seq protocol to identify TSSs that are supported by transcription, and determined that TSSs lie more than 1 kb upstream of the previously annotated TSS for nearly one-quarter of all genes. We then investigated the changes that occur in transcriptionally engaged RNA Polymerase when dosage compensation is disrupted, and find that dosage compensation controls recruitment of RNA Polymerase to X-linked genes. GRO-seq experiments (two biological replicates) were performed in nuclei from many wild-type states and a dosage compensation mutant

2013-06-18 | E-GEOD-43084 | biostudies-arrayexpress

RIp-chip experiments with the Scw1, Zfs1 and Red1 RNA-binding proteins

Project description:We have used RIp-chip to identify mRNAs that coprecipitae with specific RNA-binding proteins

2014-11-06 | E-MTAB-2709 | biostudies-arrayexpress

Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing

Project description:We developed a method, ChIP-sequencing (ChIP-seq), combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing to identify mammalian DNA sequences bound by transcription factors in vivo. We used ChIP-seq to map STAT1 targets in interferon stimulated and unstimulated human HeLa S3 cells, and compared the method's performance to ChIP-PCR and to ChIP-chip for four chromosomes. By ChIP-seq, using 15.1 and 12.9 million uniquely mapped sequence reads, and an estimated false discovery rate of less than 0.001, we identified 41,582 and 11,004 putative STAT1-binding regions in stimulated and unstimulated cells, respectively. Of the 34 loci known to contain STAT1 interferon-responsive binding sites, ChIP-seq found 24 (71%). ChIP-seq targets were enriched in sequences similar to known STAT1 binding motifs. Comparisons with two ChIP-PCR data sets suggested that ChIP-seq sensitivity was between 70% and 92% and specificity was at least 95%. Use ChIP-seq to map STAT1 targets in interferon-gamma (IFN-gamma)-stimulated and unstimulated human HeLa S3 cells, and compared the method's performance to ChIP-PCR and to ChIP-chip for four chromosomes

2009-03-23 | E-GEOD-15353 | biostudies-arrayexpress

Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization

Project description:Though important for gene regulation most studies of genome organisation use either fluorescence in situ hybridisation (FISH) or chromosome conformation capture (3C) methods. FISH directly visualises the spatial relationship of sequences, but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde crosslinking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organisation, but this has not been tested extensively. We investigate the murine HoxD locus with 5C and FISH in different developmental and activity states, and in the presence or absence of epigenetic regulators. We identify situations where the two datasets are concordant, but find other conditions where chromatin topographies extrapolated from 5C or FISH data are not compatible. We conclude that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart – influenced by nuclear environment and chromatin composition. We conclude that results obtained at high-resolution with either 3C methods or by FISH alone must be interpreted with caution and that conclusions about genome organisation should be validated by independent methods. 5C oligonucleotides were designed around EcoRI restriction sites following an alternative scheme

2015-01-02 | E-GEOD-61814 | biostudies-arrayexpress

Condensin Controls Recruitment of RNA Polymerase II to Achieve X-Chromosome Dosage Compensation

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2013-06-18 | E-GEOD-43087 | biostudies-arrayexpress

Profiling of epigenetic and transcriptomic landscapes in normal mouse liver, phenobarbital exposed mouse livers and mouse liver tumours

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2016-05-17 | E-GEOD-77731 | biostudies-arrayexpress

Effect of a KRAS peptide nucleic acid clamp on the Ion Torrent sequencing protocol

Project description:To test whether the addition of a peptide nucleic acid (PNA) clamp, which binds WT KRAS at codon 12, can increase the efficacy of mutation detection for KRASG12D within a targeted NGS setting. We tested the effect of clamping the wild-type KRAS sequence in a reference standard (Tru-Q 7, 1.3% Tier from Horizon Diagnostics, Cambridge, UK) with a KRAS c.35G>A mutation (KRASG12D) at an allelic frequency (AF) of 1.3% assessed by digital droplet PCR (ddPCR). We then re-tested the PNA on circulating-free DNA from a patient harbouring a KRASG12D mutation (at an AF of 3.2%, determined by ddPCR). Multiple runs were conducted using 10, 5, 2.5 and 1ng of DNA input.

2017-07-29 | E-MTAB-5718 | biostudies-arrayexpress

RNA-seq of the human breast cancer ERÎ±-suppressed MCF-7(MCF-7/SP10+) cells and of their internal control MCF-7 (MCF-7/C) cells

Project description:Estrogens receptor a (ERÎ±) is essential for breast tumors,since about seventy percent of breast cancers are detected as ERÎ± positive.Recent studies suggest that ERÎ± is related with the epithelial cell morphology. Recently, it has demonstrated that the suppression of ERÎ± induced epithelial-mesenchymal transition (EMT) in the MCF-7 breast cacner cells. Interestingly, the loss of ERa resulted in strong differences on the gene expression profile of a variety of genes. Therefore, the aim of the RNA-seq is to elucidate the effect of the silencing of ERÎ± on the mRNA levels of a larger variety of genes, thus revealing possible target genes which may be implicated on the aggressive phenotype and behavior of the ERÎ±-suppresed MCF-7/SP10+ breast cancer cells. For this reason total RNA from both MCF-7/SP10+ cells and of their internal control MCF-7/C cells was extracted in 3 biological replicates and 3 technical replicates.

2018-10-22 | E-MTAB-5537 | biostudies-arrayexpress

Towards understanding male infertility after spinal cord injury through quantitative proteomics

Project description:The study of male infertility after spinal cord injury (SCI) has enhanced the understanding of seminal plasma (SP) as an important regulator of spermatozoa function. However, the most important factors leading to the diminished sperm motility and viability observed in semen of men with SCI remained unknown. Thus, to explore SP related molecular mechanisms underlying infertility after SCI we used mass spectrometry-based quantitative proteomics to compare SP retrieved from SCI patients to normal controls. As a result, we present an in-depth characterization of the human SP proteome, identifying ~2,800 unique proteins, and describe, in detail, the differential proteome observed in SCI. Our analysis demonstrates that a hyper-activation of the immune system may influence some seminal processes, which probably are not triggered by microbial infection. Moreover, we show evidence of an important prostate gland functional failure, i.e. diminished abundance of metabolic enzymes related to ATP turnover, secreted via prostasomes and identify the main outcome related to this fact and that it is intrinsically linked to the low sperm motility in SCI. Together, our data suggest the molecular pathways hindering fertility in SCI and shed new light on other causes of male infertility.

2016-02-01 | PXD002145 | Pride

A high resolution map of a cyanobacterial transcriptome

Project description:Previous molecular and mechanistic studies have identified several principles of prokaryotic transcription, but less is known about the global transcriptional architecture of bacterial genomes. Here we perform a comprehensive study of a cyanobacterial transcriptome, that of Synechococcus elongatus PCC 7942, generated by combining three high-resolution data sets: RNA sequencing, tiling expression microarrays, and RNA polymerase chromatin immunoprecipitation (ChIP) sequencing. We report absolute transcript levels, operon identification, and high-resolution mapping of 5' and 3' ends of transcripts. We identify several interesting features at promoters, within transcripts and in terminators relating to transcription initiation, elongation, and termination. Furthermore, we identify many putative non-coding transcripts. We provide a global analysis of a cyanobacterial transcriptome. Our results uncover insights that reinforce and extend the current views of bacterial transcription. RNA Sequencing of the cyanobacterium Synechococcus elongatus PCC 7942 RNA polymerase ChIP Sequencing of the cyanobacterium Synechococcus elongatus PCC 7942 Tiling Microarray of the cyanobacterium Synechococcus elongatus PCC 7942

2011-05-16 | E-GEOD-29264 | biostudies-arrayexpress

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