Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

ChIP-seq mapping of histone modifications in CD14++ CD16- monocytes from human blood samples

ABSTRACT: Chromatin immunoprecipitation in combination with a genome-wide analysis via high-throughput sequencing is the state of the art method to gain genome-wide representation of histone modification or transcription factor binding profiles. However, chromatin immunoprecipitation analysis in the context of human experimental samples is limited, especially in the case of blood cells. The typically extremely low yields of precipitated DNA are usually not compatible with library amplification for next generation sequencing. We developed a highly reproducible protocol to present a guideline from the first step of isolating monocytes from a blood sample to analyse the distribution of histone modifications in a genome-wide manner. ChIP-seq histone modifications in CD14++ CD16- monocytes from human blood samples

ORGANISM(S): Homo sapiens

SUBMITTER: Marek Bartkuhn

PROVIDER: E-GEOD-54634 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

From human monocytes to genome-wide binding sites--a protocol for small amounts of blood: monocyte isolation/ChIP-protocol/library amplification/genome wide computational data analysis.

Weiterer Sebastian S Uhle Florian F Bhuju Sabin S Jarek Michael M Weigand Markus A MA Bartkuhn Marek M

PloS one 20140414 4

<h4>Unlabelled</h4>Chromatin immunoprecipitation in combination with a genome-wide analysis via high-throughput sequencing is the state of the art method to gain genome-wide representation of histone modification or transcription factor binding profiles. However, chromatin immunoprecipitation analysis in the context of human experimental samples is limited, especially in the case of blood cells. The typically extremely low yields of precipitated DNA are usually not compatible with library amplif ...[more]

PMID: 24732314

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Project description:To address the need to study frozen clinical specimens using next-generation RNA, DNA, chromatin immunoprecipitation (ChIP) sequencing and protein analyses, we developed a biobank work flow to prospectively collect biospecimens from patients with renal cell carcinoma (RCC). We describe our standard operating procedures and work flow to annotate pathologic results and clinical outcomes. We report quality control outcomes, nucleic acid yields of our RCC submissions (N=16) to The Cancer Genome Atlas (TCGA) project, as well as newer discovery platforms by describing mass spectrometry analysis of albumin oxidation in plasma and 6 ChIP sequencing libraries generated from nephrectomy specimens after histone H3 lysine 36 trimethylation (H3K36me3) immunoprecipitation. From June 1, 2010, through January 1, 2013, we enrolled 328 patients with RCC. Our mean (SD) TCGA RNA integrity numbers (RINs) were 8.1 (0.8) for papillary RCC, with a 12.5% overall rate of sample disqualification for RIN <7. Banked plasma had significantly less albumin oxidation (by mass spectrometry analysis) than plasma kept at 25°C (P<.001). For ChIP sequencing, the FastQC score for average read quality was at least 30 for 91-95% of paired-end reads. In parallel, we analyzed frozen tissue by RNA sequencing and after genome alignments, only 0.2-0.4% of total reads failed the default quality check steps of Bowtie2, which was comparable to the disqualification ratio (0.1%) of the 786-O RCC cell line, prepared under optimal RNA isolation conditions. The overall correlation coefficients for gene expression between the Mayo Clinic vs. TCGA tissues ranged from 0.75 to 0.82. These data support the generation of high-quality nucleic acids for genomic analyses from banked RCC. Importantly, the protocol does not interfere with routine clinical care. Collections over defined time points during disease treatment further enhance collaborative efforts to integrate genomic information with outcomes. Examination of H3K36me3 in ccRCC

Dataset Information

ChIP-seq mapping of histone modifications in CD14++ CD16- monocytes from human blood samples

Publications

From human monocytes to genome-wide binding sites--a protocol for small amounts of blood: monocyte isolation/ChIP-protocol/library amplification/genome wide computational data analysis.

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