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Quantifying ChIP-seq data: a spiking method providing an internal reference for sample-to-sample normalization.

ABSTRACT: Purpose: Description of a spike-adjusting-method (SAM) to normalize ChIP-seq data . Methods: We performed ChIP-seq of POLR3D and POLR2B with mouse liver supplemented with 2.5% of human DNA. Human DNA will be used as an internal control for ChIP-seq quantification. Results: We show that using the SAM for ChIP-seq quantification improve similarity of POLR3D and POLR2B ChIP-seq replicates samples and improve difference between samples originate from different conditions. Conclusions: The SAM improves comparison of ChIP-seq samples, either by increasing similarity between replicates or by emphasise differences between conditions.

ORGANISM(S): Mus musculus

PROVIDER: GSE52049 | GEO | 2014/04/02

SECONDARY ACCESSION(S): PRJNA226591

REPOSITORIES: GEO

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Quantifying ChIP-seq data: a spiking method providing an internal reference for sample-to-sample normalization.

Project description:Purpose: Description of a spike-adjusting-method (SAM) to normalize ChIP-seq data . Methods: We performed ChIP-seq of POLR3D and POLR2B with mouse liver supplemented with 2.5% of human DNA. Human DNA will be used as an internal control for ChIP-seq quantification. Results: We show that using the SAM for ChIP-seq quantification improve similarity of POLR3D and POLR2B ChIP-seq replicates samples and improve difference between samples originate from different conditions. Conclusions: The SAM improves comparison of ChIP-seq samples, either by increasing similarity between replicates or by emphasise differences between conditions. Chromatin Immuno-precipitations were performed with antibodies directed against POLR3D (Pol III) and POLR2B (Pol II) using mouse liver material supplemented with human DNA. Immuno-precipitated DNA was next sequenced using Illumina HiSeq. Three different concentrations of human spiked DNA were tested for the Pol III ChIP (2.5%, 5% and 10%). We also sequenced the corresponding inputs (crosslinked DNA from mouse liver). Two concentrations of human spiked DNA (5% and 10%) were tested for the Pol 2 ChIP. We also sequenced the corresponding inputs (crosslinked DNA from mouse liver).

2014-04-02 | E-GEOD-52049 | biostudies-arrayexpress

The effect of sample splitting and technical replication on mRNA-seq and sRNA-seq quantification

Project description:RNA sequencing (RNA-seq) is a widely used method for quantifying RNA levels across the environmental, biological and medical sciences. The accuracy of the output from an RNA-seq experiment is known to vary due to sequencing biases or errors in quantification. These have the potential to lead to false calls of differential expression (DE), and hence, to affect the accuracy of the biological inference. A proposed solution to reduce the number of false positives and increase confidence in the quality of results from such experiments is to increase the number of biological replicates. In addition, more recent suggestions are to create additional technical replicates within biological replicates (i.e. to split samples across sequencing lanes). The optimal strategy for analysing and normalizing such data, and for maximising accuracy as a function of cost, biological and technical replication is important to understand, yet currently unclear. The aim of this study was to test the effect of technical replication and sample splitting on the overall outcome of gene expression profiling for RNA-seq data.

2018-12-15 | GSE85806 | GEO

RNA-seq analysis of two genealogically linked South African Diuraphis noxia biotypes

Project description:RNA was extracted from all instar (insect developmental) stages for both D. noxia biotypes SA1 and SAM with the purpose to capture as many expressed transcripts as possible. South African D. noxia biotype SA1 is known to be the least virulent aphid, while its offspring, the South African D. noxia biotype SAM is the most virulent. The overall purpose of the experiment was to establish a baseline availability of transcripts to the aphids as well as help improve on current genome assemblies. Three biological replicates of 100 aphids each was collected from both biotypes SA1 and SAM that were respectively reared on preference host cultivars. Whole aphids were flash frozen in liquid nitrogen, ground to a powder with micro pistils and RNA was extracted making use of a Qiagen RNeasy kit. Library preparation for sequencing was performed using an Illumina TruSeq Stranded mRNA LT Sample Prep Kit following the TruSeq Stranded mRNA Sample Preparation Guide, Part # 15031047 Rev. E protocol. The replicate samples from the SAM biotype yielded between 120 – 140 million 100bp PE reads and the replicate samples from the SA1 biotype yielded between 113 – 137 million 100bp PE reads (with a Q20 phred score above 98% for all replicates) after sequencing on the NovaSeq6000 system. De novoassembly was performed making use of the Trinity package.

2020-12-31 | GSE143502 | GEO

Nrf2 Knockout vs. Wildtype mice, liver sample

Project description:In situ synthesized oligo arrays, U74Av2, from Affymetrix were used to measure differential gene expression in RNA samples generated from the liver of Nrf2 knockout and wildtype mice at 5 month age. Total RNAs from two Nrf2 knockout or wildtype littermates were analyzed separately. There are two replicates (GSM 13431, 13435) for the female Nrf2 wildtype group, two replicates (GSM 13439, 13441) for the male Nrf2 wildtype group, two replicates (GSM 13436, 13437) for the female Nrf2 knockout group, and two replicates (GSM 13438, 13440) for the male Nrf2 knockout group. Keywords: parallel sample

2003-12-16 | GSE864 | GEO

High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip

Project description:Development and evaluation of nested nanowell (N2) chip in single-cell proteomics. The newly designed chip helps to improve the sensitivity and robustness of the tandem mass tag labeling approach by sizing down wells.

2021-02-04 | MSV000086809 | MassIVE

Comparison of gene expression profile of Ruditapes philippinarum sampled in Marghera and Alberoni

Project description:Digestive Gland Samples: A manila clam oligo microarray platform (GPL10900) was used to profile gene expression in digestive gland of R. philippinarum. Total RNA was extracted from three (3) independent biological replicates of digestive gland for each sampling site, each consisting of tissue pools of five (5) animals. Statistical analysis with SAM (Significance Analysis of Microarray) identified1,127 probes differentially expressed. Gills Samples: A manila clam oligo microarray platform (GPL10900) was used to profile gene expression in gills of R. philippinarum. Total RNA was extracted from three (3) independent biological replicates of gills for each sampling site, each consisting of tissue pools of five (5) animals. Statistical analysis with SAM (Significance Analysis of Microarray) identified1,127 probes differentially expressed.

2012-08-25 | GSE27194 | GEO

Nrf2 Knockout vs. Wildtype mice, spleen sample

Project description:In situ synthesized oligo arrays, U74Av2, from Affymetrix were used to measure differential gene expression in RNA samples generated from the spleen of Nrf2 knockout and wildtype female mice at 5 month age. Total RNAs from two Nrf2 knockout or wildtype littermates were analyzed separately. There are two replicates (GSM 13470, 13472) for the female Nrf2 wildtype group and two replicates (GSM 13467, 13469) for the female Nrf2 knockout group. Keywords: parallel sample

2003-12-16 | GSE865 | GEO

H3K27me3 in four squamous cell carcinoma cell lines

Project description:ChIP-Seq for H3K27 trimethylation was performed for two HPV-positive and two HPV-negative squamous cell carcinoma cell lines. The data served two purposes. First, the data were used as an example implementation of our novel ChIP-Seq Peak Prioritization pipeline, PePr. We have developed the PePr pipeline, a ChIP-Seq Peak Prioritization pipeline that accounts for the variation among replicates and peak location relative to a gene. We show, using a transcription factor dataset (which exhibited small variation among samples), that PePr performs favorably compared to commonly used peak callers and that it achieves balanced sensitivity and specificity. We also show, using histone modification data (which exhibited larger variation among samples), that PePr can improve the detection of differential H3K27me3 regions compared with a common current approach. Using data from ChIP-Seq and gene expression experiments performed in parallel on the same samples, we show that the incorporation of functional annotations can improve the prioritization of functional sites. Secondly, the data were used to assess real differences in the genome-wide H3K27me3 profiles between HPV-positive and HPV-negative carcinoma cell lines. Careful analysis and integration of the data with DNA methylation and gene expression data performed on the same cell lines demonstrated striking differences exist.

2014-05-20 | GSE38629 | GEO

Adaptations in metabolism and protein translation give rise to the Crabtree effect in yeast

Project description:We have studied Schizosaccharomyces pombe, Saccharomyces cerevisiae, Scheffersomyces (Pichia) stipites, and Kluyveromyces marxianus yeast using the modified IBAQ label-free method with the spiked universal protein standard (UPS2). Each species had been prepared in 3 culturing replicates, and the similarity of the replicates was assessed using the TMT relative quantification methodology.

2021-11-29 | PXD026313 | Pride

A multi-omics dataset of heat-shock response in the yeast RNA binding protein Mip6 (NMR assay)

Project description:<p>Gene expression is a biological process regulated at different molecular levels, including chromatin accessibility, transcription, and RNA maturation and transport. In addition, these regulatory mechanisms have strong links with cellular metabolism. Here we present a multi-omics dataset that captures different aspects of this multi-layered process in yeast. We obtained RNA-seq, metabolomics, and H4K12Ac ChIP-seq data for wild-type and mip6delta strains during a heat-shock time course. Mip6 is an RNA-binding protein that contributes to RNA export during environmental stress and is informative of the contribution of post-transcriptional regulation to control cellular adaptations to environmental changes. The experiment was performed in quadruplicate, and the different omics measurements were obtained from the same biological samples, which facilitates the integration and analysis of data using covariance-based methods. We validate our dataset by showing that ChIP-seq, RNA-seq and metabolomics signals recapitulate existing knowledge about the response of ribosomal genes and the contribution of trehalose metabolism to heat stress.</p>

2020-02-14 | MTBLS1320 | MetaboLights

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