Universal NicE-seq for high resolution accessible chromatin profiling for native and formaldehyde fixed cells and tissues
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ABSTRACT:
PROVIDER: PRJNA589075 | ENA |
REPOSITORIES: ENA
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Project description:Accessible chromatin plays a central role in gene expression and chromatin architecture. Current accessible chromatin approaches depend on limited digestion/cutting and pasting adaptors at the accessible DNA, thus requiring additional materials and time for optimization. Universal NicE-seq (UniNicE-seq) is an improved accessible chromatin profiling method that negate the optimization step and is suited to a variety of mammalian cells and tissues. Addition of 5-methyldeoxycytidine triphosphate during accessible chromatin labeling and an on-bead library making step substantially improved the signal to noise ratio while protecting the accessible regions from repeated nicking in cell lines, mouse T cells, mouse kidney, and human frozen and FFPE tissue sections. These refinements allowed reliable mapping of accessible chromatin for high resolution genomic feature studies.
2020-10-02 | GSE140276 | GEO
Project description:Accessible chromatin landscape allows remodeling of transcription factors and enhancers during development and molecular subtypes of cancer in patient samples. One-tube universal NicE-seq (OuNicE-seq) is an improved accessible chromatin profiling method that negate DNA purification and incorporate sonication free enzymatic fragmentation before library preparation and is suited to a variety of mammalian cells. OuNicE-seq is versatile, capable of profiling 4% formaldehyde fixed chromatin in 25 fixed cells and opens the possibility for automation. Accessible chromatin profile is more efficient on formaldehyde fixed cells using OuNicE-seq compare to Tn5 transposon mediated methods, demonstrating its versatility.
2021-12-01 | GSE175651 | GEO
Project description:Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize cellular subpopulations and great advances have been made using this approach. Cells are often fixed with formaldehyde prior to the sorting process to preserve cell morphology and maintain the expression of surface molecules, as well as to ensure safety in the sorting of infected cells. It is widely recognized that formaldehyde fixation alters RNA and DNA structure and integrity, thus analyzing gene expression in these cells has been difficult. We therefore examined the effects of formaldehyde fixation on the stability and quantitation of nucleic acids in cell lines, primary leukocytes and also cells isolated from SIV-infected pigtailed macaques. We developed a method to extract RNA from fixed cells that yielded the same amount of RNA as our common method of RNA isolation from fresh cells. Quantitation of RNA by RT-qPCR in fixed cells was not always comparable with that in unfixed cells. In comparison, when RNA was measured by the probe-based NanoString system, there was no significant difference in RNA quantitation. In addition, we demonstrated that quantitation of proviral DNA in fixed cells by qPCR is comparable to that in unfixed cells when normalized by a single-copy cellular gene. These results provide a systematic procedure to quantitate gene expression in cells that have been fixed with formaldehyde and sorted by FACS.
2013-09-09 | GSE50690 | GEO
Project description:In crosslinked MINUTE-ChIP, formaldehyde-fixed chromatin is fragmented using sonication, blunted and ligated to double-stranded DNA adaptors that include a T7 RNA polymerase promoter and a sample barcode sequence. Finally, samples are combined and subsequent ChIP reactions are performed with the pooled samples. ChIP material is prepared into an Illumina-compatible library using linear amplification by virtue of T7 RNA polymerase, reverse transcription and a low-cycle library PCR amplification. Here, we demonstrate a MINUTE-ChIP for Nanog from formaldehyde-fixed cells using fragmentation by sonication.
2024-02-11 | GSE244908 | GEO
Project description:Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize cellular subpopulations and great advances have been made using this approach. Cells are often fixed with formaldehyde prior to the sorting process to preserve cell morphology and maintain the expression of surface molecules, as well as to ensure safety in the sorting of infected cells. It is widely recognized that formaldehyde fixation alters RNA and DNA structure and integrity, thus analyzing gene expression in these cells has been difficult. We therefore examined the effects of formaldehyde fixation on the stability and quantitation of nucleic acids in cell lines, primary leukocytes and also cells isolated from SIV-infected pigtailed macaques. We developed a method to extract RNA from fixed cells that yielded the same amount of RNA as our common method of RNA isolation from fresh cells. Quantitation of RNA by RT-qPCR in fixed cells was not always comparable with that in unfixed cells. In comparison, when RNA was measured by the probe-based NanoString system, there was no significant difference in RNA quantitation. In addition, we demonstrated that quantitation of proviral DNA in fixed cells by qPCR is comparable to that in unfixed cells when normalized by a single-copy cellular gene. These results provide a systematic procedure to quantitate gene expression in cells that have been fixed with formaldehyde and sorted by FACS. We compared RNA quantitation between unfixed and formaldehyde-fixed cells using the NanoString nCounter system. This analysis was performed using two cell lines, K562 and CEMx174, and human PBMCs. Three biological replicates were performed for each cell type. We also performed this comparison using human PBMCs sorted by FACS. For this analysis, we isolated PBMCs from two donors and performed two technical replicates for each donor sample.
2013-09-09 | E-GEOD-50690 | biostudies-arrayexpress
Project description:The "Assay for Transposase Accessible Chromatin sequencing" (ATAC-seq) is an efficient and easy to implement protocol to measure chromatin accessibility that has been widely used in multiple applications studying gene regulation. While several modifications or variants of the protocol have been published since it was first described, there has not yet been an extensive evaluation of the effects of specific protocol choices head-to-head in a consistent experimental setting. In this study, we tested multiple protocol options for major ATAC-seq components (including three reaction buffers, two reaction temperatures, two enzyme sources, and the use of either native or fixed nuclei) in a well-characterized cell line. In addition, the native conditions were tested in a primary sample type (mouse lung tissue) with two different input amounts. In general, native samples yielded more peaks (particularly at loci not overlapping transcription start sites) than fixed samples, and the temperature at which the enzymatic reaction was carried out had a major impact on data quality metrics for both fixed and native nuclei. However, the effect of various conditions tested was not always consistent between the native and fixed samples. For example, the Nextera and Omni buffers were largely interchangeable across all other conditions, while the THS buffer resulted in markedly different profiles in native samples. In-house and commercial enzymes performed similarly. We found that the relationship between commonly used measures of library quality differed across temperature and fixation, and so evaluating multiple metrics in assessing the quality of a sample is recommended. Notably, we also found that these choices can bias the functional class of elements profiled and so we recommend evaluating several formulations in any new experiments. Finally, we hope the ATAC-seq workflow formulated in this study on crosslinked samples will help to profile archival clinical specimens.
2022-03-23 | GSE174280 | GEO
Project description:Extensive evaluation of ATAC-seq protocols for native or formaldehyde-fixed nuclei
| PRJNA729079 | ENA
Project description:Accessible chromatin plays a central role in gene expression and chromatin architecture. Current accessible chromatin approaches depend on limited digestion/cutting and pasting adaptors at the accessible DNA, thus requiring additional materials and time for optimization. Universal NicE-seq (UniNicE-seq) is an improved accessible chromatin profiling method that negates the optimization step and is suited to a variety of mammalian cells and tissues. Addition of 5-methyldeoxycytidine triphosphate during accessible chromatin labeling and an on-bead library making step substantially improved the signal to noise ratio while protecting the accessible regions from repeated nicking in cell lines, mouse T cells, mouse kidney, and human frozen tissue sections. We also demonstrate one tube UniNicE-seq for the FFPE tissue section for direct NGS library preparation without sonication and DNA purification steps. These refinements allowed reliable mapping of accessible chromatin for high-resolution genomic feature studies.
| S-EPMC7507628 | biostudies-literature
Project description:One-pot universal NicE-seq: All enzymatic downstream processing of 4% formaldehyde crosslinked cells for chromatin accessibility genomics
| PRJNA733150 | ENA
Project description:Formaldehyde-fixed RNA-Immunoprecipitation (fRIP for heterogenous ribonucleoprotein K (hnRNP K)
| PRJNA522053 | ENA