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ABSTRACT:
PROVIDER: PRJNA732942 | ENA |
REPOSITORIES: ENA
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Project description:Single fluorescent protein biosensor engineering
| PRJNA324720 | ENA
Project description:The ability to analyze chromatin accessibility at the single-nucleus level is a critical step towards capturing the highly complex mechanisms that regulate gene expression in the human brain, and to functionally characterize the genetic architecture of neuropsychiatric traits. The assay for transposase accessible chromatin (ATAC-seq) has rapidly established itself as a robust means of studying epigenome regulation in bulk tissues and, more recently, at the single-cell level. To facilitate optimal preparation of frozen archival human brain tissue samples for single-nuclear ATAC-seq (snATAC-seq), we assessed the impact of different sample preparation approaches on the quality of snATAC-seq libraries. We tested six conditions, including different fluorescent activated nuclear sorting (FANS) strategies, from nuclei isolated from a sample of human cortical tissue. Using an unbiased computational approach, we were able to identify major cell populations in all snATAC-seq libraries, including those not subjected to FANS. When FANS was used, we observed that the choice of DNA stain impacts the quality of ATAC-seq libraries. Staining samples with DAPI can lead to insufficient detection of open chromatin regions, whereas 7aad provides improved identification of some nuclear sub-populations. We also observed that use of fluorescent dyes (in particular, DAPI) can lead to under-sampling of excitatory neurons. Our work provides guidelines on the trade-offs associated with different nuclear isolation strategies when preparing samples for snATAC-seq.
2022-01-29 | GSE166016 | GEO
Project description:We used mice that conditionally expressed a fluorescent reporter only in TMEM119 expressing cells to sort microglia for RNA-sequencing. We then performed gene expression profiling analysis using data obtained from RNA-seq of three different brain regions (prefrontal cortex, striatum, midbrain) in both male and female mice.
2022-07-27 | GSE203553 | GEO
Project description:To demonstrate our method of controlling for technical noise in single-cell RNA-seq experiments we manually collected single A. thaliana cells marked by the expression of green fluorescent protein (GFP) driven by either the GL2 or WOX5 promoters. Seven and six cells were collected from each cell type, respectively. The GL2 promoter marks the non-hair cells in the root epidermis whereas the WOX5 promoter specifies the quiescent center (QC) of the root. Each cell selected was processed together with 50 pg of total HeLa RNA spike-in to prepare RNA-seq libraries using the Tang protocol. For comparison, we again added the commercially available ERCC spike-ins. We also performed RNA-seq on a set of technical replicates of total A. thaliana RNA using starting amounts ranging from 5000pg down to 10pg, a range that covers the RNA content obtainable from single cells of various sizes.
2013-03-21 | E-MTAB-1499 | biostudies-arrayexpress
Project description:genotypes of fluorescent protein variants
| PRJNA540481 | ENA
Project description:genotypes of fluorescent protein variants
| PRJNA631354 | ENA
Project description:We report the genomic sequences bound by the C2H2 transcription factors BLUEJAY and JACKDAW as profiled by chromating immunoprecipitation experiments followed by Illumina high sequencing. Plants were carrying constructs of proteins (translational) fusions of these transcription factors to the yellow fluorescent protein. Gene fusions were expressed under all their genomic regulatory regions using recombineering. The recombineering technique was used to recombine the fluorecent protein at the 3' end of their genomic regions in a JATY clone carrying these genes. Subsequently the JATY clones carrying these translational fusions were introgressed into plants. We performed ChIP on roots grown under standard conditions and fixed with formaldehyde in PBS-EDTA. Sequences bound by BLUEJAY and JACKDAW were immunoprecipitated with a-GFP AbCam290 at 4 °C overnight. Biological replicate samples were make into libraries of ~100 bp insert size and 50 bp of single ends sequenced through Illumina High Sequencing. Two biological replicates of samples of plants expressing the C2H2 transcription factors fused to the yellow fluorescent protein; and of control wild type plants processed through the same protocol.
2015-11-02 | E-GEOD-60011 | biostudies-arrayexpress
Project description:The lack of tools to monitor the dynamics of (pseudo)hypohalous acids in live cells and tissues hinders a better understanding of inflammatory processes. Here we present a fluorescent genetically encoded biosensor, Hypocrates, for the visualization of (pseudo)hypohalous acids and their derivatives. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from Escherichia coli. We show that Hypocrates is ratiometric, reversible, and responds to its analytes in the 106 M-1s-1 range. Solving the Hypocrates X-ray structure provided insights into its sensing mechanism, making it the first circularly permuted fluorescent protein-based redox probe for which spatial organization is determined. We exemplify its applicability by imaging hypohalous stress in bacteria phagocytosed by primary neutrophils. Finally, we demonstrate that Hypocrates can be utilized in combination with HyPerRed for the simultaneous visualization of (pseudo)hypohalous acids and hydrogen peroxide dynamics in a zebrafish tail fin injury model
2021-12-15 | PXD029624 | Pride
Project description:Examination of EBf1 binding by ChIP-seq in differentiated human adipose stromal cell (hASC) pre-adipocyte Pre-Adipocytes differentiated in-vitro were fixed in 1% formaldehyde for 15 min at room temperature and quenched for 5 min by adding glycine to a final concentration of 0.125 M. ChIP assays were then performed with custom-made EBF1 antibody or rabbit IgG . ChIP-sequencing libraries were prepared using NEBnext chip-seq library Prep master mix set from 5 ng of anti-EBF1 and anti-IgG ChIP DNA, respectively. Sequence data were generated with Illumina HiSeq 2000 single-read sequencing and aligned against the human genome (hg19, NCBI).
2014-05-22 | E-GEOD-54889 | biostudies-arrayexpress