Systematic evaluation of DNase-seq experimental parameters
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Housheng He
PROVIDER: E-GEOD-51915 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:We systematicaly evaluated two major factors, enzyme digestion level and fragment size, that would affect DNase-seq experiment. We found that while under- or over-digestion sigificantly decreases the DNase-seq signal, there is a broad range of suitable digestion level. In addition, we found fragment smaller than nucleosome size is optimal to identify transcription factor binding events.
2013-11-25 | GSE51915 | GEO
Project description:The experiment was designed to investigate transcriptional dynamics during definitive endoderm differentiation of human embryonic stem cells (hESCs) in early G1 phase of the cell cycle. For this, the FUCCI system was used to sort hESCs in Early G1 (EG1). Samples were taken at 0h (hESCs), 12h, 24h, 36h, 48h, 60h and 72h in triplicates. Total RNA was extracted using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) and residual genomic DNA was removed using the On-Column DNase I Digestion Set (Sigma-Aldrich) following manufacturer’s instructions. The libraries were generated at a library fragment size between 100 bp to 1 kb with Stranded RNAseq Standard with Oligo dT pulldown. All samples were amplified with a standard 10 PCR cycle before sequencing. The samples were multiplexed and analysed by Illumina Hiseq V4 with a paired end read-length PE75. RNA-seq data analysis was performed following recommendations described in Conesa et al. (DOI https://doi.org/10.1186/s13059-016-0881-8).
2020-09-07 | E-MTAB-9194 | biostudies-arrayexpress
Project description:Intact nuclei from an asynchronous population of W303 Saccharomyces cerevisiae in log-phase growth were subjected to a 16-minute DNase I digestion (0.1 U/μL) at 37 °C. DNA was then recovered, and single-end Illumina sequencing libraries were prepared using the Crawford DNase-seq method (Song and Crawford, 2010).
2016-01-08 | GSE69651 | GEO
Project description:Purpose: The goals of this study are to compare the wild type and phoU1 or phoU2 gene knock out transcriptome profiling (RNA-seq) to analysis gene expression change Methods: Samples was prepared according to the Illumina RNA Sequencing Sample Preparation Guide. In brief, three biological replicates for each of the S. aureus and derivative strains were treated with RNase-free DNase I (Takara) to remove the genomic DNA. BioAnalyzer 2100 system used to evaluate the RNA quality. Samples were treated with RiboZero rRNA removal kit (gram-positive organisms) to remove ribosomal RNA. Fragmented RNA was reverse transcribed using random primers. The cDNA library included the fragment size (200–300 bp) were prepared by the mRNA-Seq Sample Prep kit and verified on a BioAnalyzer 2100 system. Then, the fragment size is amplified by Illumina cBot and sequenced by Illumina HiSeq 2500. Quality control involves discards of rRNA reads, sequencing adapters, short-fragment and other low-quality reads. The remaining reads were multi-mapped to the genome of S. aureus USA500 2395 at the NCBI website with Bowtie2 software. BED Tools software was used to count the transcript expression levels. Per Kilobase of Gene Per Million Mapped Reads (RPKM) reported RNA-seq gene expression values. Integrated Genomics Viewer was used to visualize the date. DEGseq software was used to quantify differential expression of different transcripts. Significant differences in expression ratios were defined at least a 2.0-fold or 0.5-fold change in transcript level. Results:Transcriptome analysis revealed that 573 or 285 genes were differentially expressed by at least 2.0-fold in the ΔphoU1 or ΔphoU2 mutant’s vs the wild type. Genes involved in carbon and pyruvate metabolism were changed, and virulence genes and virulence regulatory genes were downregulated Conclusions: In conclusion, both PhoU1 and PhoU2 in S. aureus regulate virulence and persister via the metabolism.
2019-10-19 | GSE139071 | GEO
Project description:Conformational changes in proteins can lead to disease. Thus, methods for identifying conformational changes in proteins can further improve our understanding and facilitate detection of disease states. Here we combine limited proteolysis (LiP) with Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) to characterize breast cancer-related conformational changes in proteins on the proteomic scale. Studied here are the conformational properties of proteins in two cell culture models of breast cancer, including the MCF-10A and MCF-7 cell lines. The SILAC-LiP approach described here identified ~200 proteins with cell-line dependent conformational changes, as determined by their differential susceptibility to proteolytic digestion using the non-specific protease, proteinase K. The protease susceptibility profiles of the proteins in these cell lines were compared to thermodynamic stability and expression level profiles previously generated for proteins in these same breast cancer cell lines. The comparisons revealed that there was little overlap between the proteins with protease susceptibility changes and the proteins with thermodynamic stability and/or expression level changes. Thus, the large-scale conformational analysis described here provides unique insight into the molecular basis of the breast cancer phenotypes in this study.
2016-12-23 | PXD005210 | Pride
Project description:Currently, drug resistance of anti-cancer therapy has become the main cause of low survival rate and poor prognosis. Full understanding of drug resistance mechanisms is an urgent request for further development of anti-cancer therapy and improvement of prognosis. Here we present our N-glycoproteomics study of putative N-glycoprotein biomarkers of drug resistance in doxorubicin resistance breast cancer cell line michigan cancer foundation-7 (MCF-7/ADR) relative to parental michigan cancer foundation-7 (MCF-7) cells. Intact N-glycopeptides (IDs) from MCF-7/ADR and MCF-7 cells were enriched with zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC), labeled with stable isotopic diethylation (SIDE), and analyzed with C18-RPLC-MS/MS (HCD with stepped normalized collision energies); these IDs were identifed with database search engine GPSeeker, and the diferentially expressed intact N-glycopeptides (DEGPs) were quantifed with GPSeekerQuan. With target-decoy searches and control of spectrum-level FDR≤1%, 322 intact N-glycopeptides were identifed; these intact N-glycopeptides come from the combination of 249 unique peptide backbones (corresponding to 234 intact N-glycoproteins) and 90 monosaccharide compositions (corresponding to 248 putative N-glycosites). The sequence structures of 165 IDs were confrmed with structure-diagnostic fragment ions. With the criteria of observation at least twice among the three technical replicates,≥1.5-fold change and p value<0.05, 20 DEGPs were quantifed, where fve of them were up-regulated and 15 of them were down-regulated; the corresponding intact N-glycoproteins as putative markers of drug resistance were discussed.
2023-12-30 | PXD036101 | Pride
Project description:We performed microRNA sequencing (miRNA-seq) according to Illumina sequencing protocols. Total RNA extracted from MCF-7 cells expressing human ER-beta in absence of estrogen stimuli was subjected to size selection labrary preparation ond retrotranscribed. Obtained DNA was sequenced with the Illumina/Solexa Genome Analyzer (GAIIX).
2014-11-13 | E-MTAB-2085 | biostudies-arrayexpress
Project description:Gene expression is controlled by the complex interaction of transcription factors binding to promoters and other regulatory DNA elements. One common characteristic of the genomic regions associated with regulatory proteins is a pronounced sensitivity to DNase I digestion. We generated genome-wide high resolution maps of DNase I hypersensitive (DH) sites from both seedling and callus tissues of rice. Approximately 25% of the DH sites from both tissues were found in the putative promoters, indicating that the vast majority of gene regulatory elements in rice are not located at promoter regions. We found 58% more DH sites in callus than in seedling. For DH sites detected in both seedling and callus, 31% displayed significantly different levels of DNase I sensitivity within the two tissues. Genes that were differentially expressed in seedling and callus were frequently associated with DH sites in both tissues. The DNA sequences contained within the DH sites were hypomethylated, consistent with what is known about active gene regulatory elements. Interestingly, tissue-specific DH sites located in the promoters showed an elevated level of DNA methylation. A distinct elevation of H3K27me3 was associated with intergenic DH sites. These results suggest that epigenetic modifications play a role in the dynamic changes of the numbers and DNase I sensitivity of DH sites during development.
2011-11-09 | GSE26733 | GEO
Project description:Genome-wide analysis of H3K4me3 modifications, Gata1 binding, and DNase I hypersensitivity sites in zebrafish adult red blood cells Zebrafish peripheral blood nuclei were isolated for DNase I hypersensitivity assays. Total DNA were purified using the Proteinase K digestion and Phenol-Chloroform extraction. Small DNase I fragments from the DNaseI treatement were enriched using a sucrose gradient. These ends were then labeled and amplified during library construction and for Solexa sequencing
2012-02-17 | E-GEOD-35875 | biostudies-arrayexpress
Project description:Intact nuclei from an asynchronous population of W303 Saccharomyces cerevisiae in log-phase growth were subjected to a 16-minute DNase I digestion (0.1 U/μL) at 37 °C. DNA was then recovered, and single-end Illumina sequencing libraries were prepared using the Crawford DNase-seq method (Song and Crawford, 2010). Two biological replicates of DNase-seq were sequenced in single-end mode on an Illumina HiSeq 2000.
2016-01-08 | E-GEOD-69651 | biostudies-arrayexpress