Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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H3K4me3 CUT&RUN profiling in wild-type mouse embryonic stem cells (mESCs)

ABSTRACT: H3K4me3 occupancy profiles were generated for wild-type mESCs using the CUT&RUN assay. Matched non-targeting IgG samples were included as negative controls to ensure specificity of enrichment.

INSTRUMENT(S): Illumina NovaSeq 6000, deeptools, macs3

ORGANISM(S): Mus musculus

SUBMITTER: Kashyap Chhatbar

PROVIDER: E-MTAB-17000 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Hematopoiesis in embryonic and adult life is the process of producing blood cells. In mammalian embryos, hematopoiesis occurs in three consecutive overlapping waves (Neo et al. 2021; Dzierzak and Bigas 2018) and is regulated by transcription factors (TFs) and signaling molecules. In this study, we investigated the function of three relatively poorly studied TFs in early embryonic hematopoietic development at single-cell resolution: Activating transcription factor 3 (Atf3), Zinc finger protein 711 (Zfp711), and B cell CLL/lymphoma 6, member B (Bcl6b) respectively. We observed that these three TFs are upregulated early in development when hematopoietic and endothelial lineages separate from cardiac and other mesodermal lineages. To study the roles of these TFs in a rapidly changing system with diverse cell types and small cell populations during early developmental stages, we employed multiplexed single-cell RNA sequencing (scRNA-seq) of TF knockouts (KO) of in vitro differentiating mouse embryonic stem cells (mESCs) and also capturing changes with Flow Cytometric Analysis (FCA). This approach offered a valuable method to dissect the functions of these TFs in lineage induction, specification, and separation, providing access to sufficient numbers of various progenitor cells. We adapted available multiplexing technology for single-cell RNA sequencing (scRNA-seq) -multiplexing knockouts (KO) and Control conditions with biological replicates-, accompanied by Flow Cytometric Analysis (FCA) to study the role of three TFs in early embryonic hematopoietic development. Using this adaptation, the depth and coverage of this study can be placed between large-scale multiplexed CRISPR-based perturbation studies covering multiple candidate genes together (Datlinger et al. 2017; Jaitin et al. 2016; Dixit et al. 2016; Adamson et al. 2016) and single gene perturbation studies, which focus on the in-depth function/role of a particular gene with multiple experimental procedures (Harland et al. 2021). With adapted methodology, we studied the role of the three TF genes at once in a cost-efficient manner in one experiment by including three biological replicates to minimize false positive results, to capture a sufficient number of cells to detect changes in low abundance cell types, to minimize the creation of potential batch effects (e.g., each replicates creates a separate library) and prevent the loss of biological information during computational integration steps by skipping computational integration step. Following the scRNA-seq analysis, our findings are compared with publicly available datasets to categorize our findings. This categorized information can be used as a launching pad for future in-depth follow-up studies investigating the roles of these three TFs in hematoendothelial development.

Dataset Information

H3K4me3 CUT&RUN profiling in wild-type mouse embryonic stem cells (mESCs)

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