Titration-based normalization of antibody amount improves consistency of ChIP-seq experiment
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PROVIDER: PRJNA824452 | ENA |
REPOSITORIES: ENA
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Project description:Chromatin immunoprecipitation (ChIP) is an antibody-based approach that is most frequently utilized in research areas of chromatin biology and epigenetics. The ENCODE Consortium provided guidelines about antibody but the fundamental aspect regarding antibody titer was not addressed. Here, we introduced a simple and quick DNA-based quantification of chromatin input, allowing to normalize the antibody amount in individual ChIP reaction to the optimal titer. We further demonstrated the titration-based normalization of antibody amount generates improved assay outcome with high consistency among samples in an experiment or recurrent experiments for a broad range of chromatin input amount. Chromatin immunoprecipitation-sequencing (ChIP-seq) was performed for histone H3K27ac mark in the conditions of various titer of antibody in ChIP reactions..
2023-04-12 | GSE200404 | GEO
Project description:Chromatin immunoprecipitation (ChIP) is an antibody-based approach that is frequently utilized in chromatin biology and epigenetics. The challenge in experimental variability by unpredictable nature of usable input amounts from samples and undefined antibody titer in ChIP reaction still remains to be addressed. Here, we introduce a simple and quick method to quantify chromatin inputs and demonstrate its utility for normalizing antibody amounts to the optimal titer in individual ChIP reactions. For a proof of concept, we utilized ChIP-seq validated antibodies against the key enhancer mark, acetylation of histone H3 on lysine 27 (H3K27ac), in the experiments. The results indicate that the titration-based normalization of antibody amounts improves assay outcomes including the consistency among samples both within and across experiments for a broad range of input amounts.
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2014-07-24 | E-GEOD-49609 | biostudies-arrayexpress
Project description:In order to test the transcriptome-wide functionality of the identified sRSE instances, we performed an in vivo titration experiment in which synthetic RNA oligonucleotides harboring tandem sRSE1 repeats were used as intracellular decoys that would bind the putative trans factor, preventing it from targeting endogenous transcripts.
2014-07-24 | GSE49609 | GEO
Project description:Comparison to RNA-Seq showed different strengths and weaknesses for different regimes of expression strength. At sufficient read-depth, both platforms seemed comparable for gene-level expression profiling. Comparison to RNA-Seq showed comparable accuracy and precision with microarrays for ERCC spike-ins at medium to high expression levels. At low expression levels, the array showed signal attenuation but better precision, while RNA-Seq maintained accuracy albeit with highly inflated variance. In summary, both platforms can be meaningfully applied in a similar range of expression strength. Assessment of (precision, accuracy, reproducibility, mutual information, titration order consistency, and known mixing ratio recovery) by measurement of known-ratio mixtures of two RNA reference samples. The array probes 776 complex genes representative of the AceView gene model annotation, as well as 92 ERCC spike-in controls.
2015-04-03 | E-GEOD-48016 | biostudies-arrayexpress
Project description:Illumina Infinium whole genome genotyping (WGG) arrays are increasingly being applied in cancer genomics to study gene copy number alterations and allele-specific aberrations such as loss-of-heterozygosity (LOH). Methods developed for normalization of WGG arrays have mostly focused on diploid, normal samples. However, for cancer samples genomic aberrations may confound normalization and data interpretation. Therefore, we examined the effects of the conventionally used normalization method for Illumina Infinium arrays when applied to cancer samples. We demonstrate an asymmetry in the detection of the two alleles for each SNP, which deleteriously influences both allelic proportions and copy number estimates. The asymmetry is caused by a remaining bias between the two dyes used in the Infinium II assay after using the normalization method in Illumina’s proprietary software (BeadStudio). We propose a quantile normalization strategy for correction of this dye bias. We tested the normalization strategy using 535 individual hybridizations from 10 data sets from the analysis of cancer genomes and normal blood samples generated on Illumina Infinium II 300k version 1 and 2, 370k and 550k BeadChips. We show that the proposed normalization strategy successfully removes asymmetry in estimates of both allelic proportions and copy numbers. Additionally, the normalization strategy reduces the technical variation for copy number estimates while retaining the response to copy number alterations. The proposed normalization strategy represents a valuable low-level analysis tool that improves the quality of data obtained from Illumina Infinium arrays, in particular when used for LOH and copy number variation studies.
2008-10-04 | GSE11977 | GEO
Project description:Two samples with known methylation differences. Sample A is NA 10923 from Coriell Institute for Medical Research. It is a B-Lymphocyte sample from a male donor. Sample B is HTB-38 cell line from ATCC (http://www.atcc.org). It is a colon cancer sample from a female donor. Sample A and B were normalized into the same concentration, and then mixed in five different titration ratios: 100:0, 90:10, 75:25, 50:50 and 0:100 Sample A and Sample B were mixed in five different titration ratios: 100:0, 90:10, 75:25, 50:50 and 0:100
2010-12-01 | E-GEOD-23789 | biostudies-arrayexpress
Project description:MNase titration-Seq of Saccharomyces cerevisiae.
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Project description:Illumina Infinium whole genome genotyping (WGG) arrays are increasingly being applied in cancer genomics to study gene copy number alterations and allele-specific aberrations such as loss-of-heterozygosity (LOH). Methods developed for normalization of WGG arrays have mostly focused on diploid, normal samples. However, for cancer samples genomic aberrations may confound normalization and data interpretation. Therefore, we examined the effects of the conventionally used normalization method for Illumina Infinium arrays when applied to cancer samples. We demonstrate an asymmetry in the detection of the two alleles for each SNP, which deleteriously influences both allelic proportions and copy number estimates. The asymmetry is caused by a remaining bias between the two dyes used in the Infinium II assay after using the normalization method in Illumina’s proprietary software (BeadStudio). We propose a quantile normalization strategy for correction of this dye bias. We tested the normalization strategy using 535 individual hybridizations from 10 data sets from the analysis of cancer genomes and normal blood samples generated on Illumina Infinium II 300k version 1 and 2, 370k and 550k BeadChips. We show that the proposed normalization strategy successfully removes asymmetry in estimates of both allelic proportions and copy numbers. Additionally, the normalization strategy reduces the technical variation for copy number estimates while retaining the response to copy number alterations. The proposed normalization strategy represents a valuable low-level analysis tool that improves the quality of data obtained from Illumina Infinium arrays, in particular when used for LOH and copy number variation studies. To investigate the effects of a quantile normalization of Illumina Infinium data, compared to conventional normalization using BeadStudio (www.illumina.com), we renormalized 535 individual hybridizations conducted on Illumina 300K, 370K and 550K BeadChips. Sample types included breast cancer, colon cancer, urothelial carcinoma, leukemia as well as normal blood and HapMap samples. This series includes the 6 breast cancers hybridized on Illumina HumanHap 550K BeadChips.
2008-10-04 | E-GEOD-11977 | biostudies-arrayexpress
Project description:Direct sequencing of low DNA amount
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