Amplification of pico-scale DNA mediated by bacterial carrier DNA for small-cell-number transcription factor ChIP-seq
ABSTRACT: ChIP-seq is used to map transcription factor occupancy and generate epigenetic profiles genome-wide. The requirement of nano-scale ChIP DNA for generation of sequencing libraries has impeded ChIP-seq on in vivo tissues of low cell numbers. We describe a robust, simple and scalable methodology for ChIP-seq of low-abundant cell populations, reliably amplifying 50 pg of ChIP DNA, corresponding to ~30,000 input cells for transcription factor ChIP (CEBPA) and 3,000 cells for histone mark ChIP (H3K27me3). This represents a significant advance compared to existing technologies, which involve complex and time-consuming steps of pre-amplification, making them susceptible to experimental biases. ChIP-seq of histone modifications H3K27me3 (2 biological replicates (I+II) , 2 ng input), H3K4me3 (2 biological replicates (II+III), 2 ng input), transcription factor CEBPA (2 biological replicates (I+II), 300 pg input), 4 diluted CEBPA libraries (pool of ChIP from 3 biol. replicates (I+II+III) 3x 100 pg input, 1x 50 pg). Additonal ChIP-seq using 10,000 cells, 1 biological replicate of each H3K4me3 and CEBPA.
Project description:This study aims to investigate whether the passage of human chromosome 21 through the mouse male germline results in changes in the transcriptional deployment of the exogenous chromosome in the offspring generation. We used the Tc1 mouse model that stably carries almost an entire copy of human chromosome 21 and profiled the genome-wide pattern of H3K4me3, H3K27ac, CEBPA, HNF4A and RNA polymerase II in liver tissue of male and female-germline derived Tc1 mice using ChIP-Seq. Furthermore, the genome-wide pattern of H3K4me3 was profiled in additional tissues including kidney, liver and brain.
Project description:We profiled the genome-wide occupancy of three tissue-specific transcription factors, HNF4A, CEBPA and FOXA1, as well as the genome-wide occurrence of the histone mark, H3K4me3 in the livers of two inbred parental mouse strains (C57BL/6J and CAST/EiJ) and their F1 crosses. We also included H3K27ac data generated from F1 hybrids as well as the profiling of HNF4A, CEBPA and FOXA1 in both CEBPA and HNF4a heterozygous knock-outs.
Project description:This study aims to study genome wide location of HNF4a, FoxA1 and CEBPA in wild type rat liver. Please note that all raw data files for this study were replaced with new versions on 12 March 2014 because the previous versions are corrupted. The corrupted files are associated with ENA run accessions ERR215698 to ERR215705 and should not be used. The correct files are associated with ENA run accessions ERR458085 to ERR458092.
Project description:Expression analysis of CEBPa knockout effects on gene expression in adult mouse liver. We used a conditional knock out strategy to delete CEBPa specifically in adult mouse hepatocytes and analysed the resulting changes in gene expression by means of microarrays.
Project description:Analysis of global RNA expression of Cebpa knockdown lineage-negative marrow cells reveals known and potentially novel C/EBPa targets. Total RNA from lineage-negative marrow progenitors transduced with vector or Cebpa shRNA and cultured under myeloid differentiation condition for 2 days were compared.
Project description:Purpose: CEBPA mutations are found as either biallelic (biCEBPA) or monoallelic (moCEBPA). We set out to explore whether the kind of CEBPA mutation is of prognostic relevance in cytogenetically normal AML (CN-AML). Patients and Methods: 467 homogeneously treated CN-AML patients were subdivided into moCEBPA, biCEBPA and wildtype (wt) CEBPA patients. The subgroups were analyzed for clinical parameters and for additional mutations in the NPM1, FLT3 and MLL genes. Furthermore, we obtained gene expression profiles (GEP) for a subgroup of 61 patients using oligonucleotide microarrays. 61 bone-marrow samples from CN-AML patients were analyzed using Affymetrix HG-U133 oligonucleotide microarrays (Affymetrix, Santa Clara, CA). Sample preparation, hybridization and image acquisition were performed according to standard Affymetrix protocols. Custom chip definition files based on the GeneAnnot database were used for data analysis (Ferrari et al, BMC Bioinformatics 8:446). The twilight algorithm was used to comapre gene expression profiles of patients with wildtype CEBPA and mono- and biallelic CEBPA mutations.
Project description:Here, we have focused on studying the link between metabolic changes driven by the differentiation into mature adipocytes of a human preadipocyte cell line (SGBS) and their regulation, through a combined experimental and computational approach. By collecting data on gene expression, PPARg, CEBPa, LXR and H3K4me3 genome-wide ChIP-seq profles and transcriptome-wide microRNA target identification for miR-27a, miR29a and miR-222, and using constraint-based modeling to estimate metabolic reaction activity, we obtained a comprehensive set of information highlighting how epigenetic, transcriptional and post-transcriptional regulation impacts the metabolic network. Illumina Solexa sequencing: Six samples in total. Two ChIP-seq samples were prepared using an antibody against H3K4me3 active TSS chromatin marker from human SGBS preadipocyte and day 10 differentiated SGBS adipocyte cells. From day 10 differentiated SGBS cells additional three samples were prepared using an antibody against PPARg, CEBPa and LXRa to determine their genome-wide binding. One input control sample is included.
Project description:Bivalent H3K4me3 and H3K27me3 chromatin domains in embryonic stem cells keep active developmental regulatory genes expressed at very low levels and poised for activation. Here, we show an alternative and previously unknown bivalent modified histone signature in lineage-committed mesenchymal stem cells and preadipocytes that pairs H3K4me3 with H3K9me3 to maintain adipogenic master regulatory genes (Cebpa and Pparg) expressed at low levels yet poised for activation when differentiation is required. We show lineage-specific gene-body DNA methylation recruits H3K9 methyltransferase SETDB1 which methylates H3K9 immediately downstream of transcription start sites marked with H3K4me3 to establish the bivalent domain. At the Cebpa locus, this prevents transcription factor C/EBPβ binding, histone acetylation, and further H3K4me3 deposition and is associated with pausing of RNA polymerase II, which limits Cebpa gene expression and adipogenesis. H3K4me3, H3K27me3, H3K9me3, SETDB1, MBD1, and Pol II ChIP-seq. m5CpG pull-down using recombinant MBD domain of MBD1 followed by next-generation sequencing.