Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Single-Cell Multi-omics RNA and ATAC Sequencing of In Vitro Hematoendothelial Differentiation from Mouse Embryonic Stem Cells

ABSTRACT: An in vitro differentiation system using mouse embryonic stem cells to capture multiple differentiation timepoints: days 3, 4, 5, 6, and 7 as scMultiome (ATAC+RNA) and days 0, 2, and 4 as scRNA-seq. scMultiome libraries were generated using the Single Cell Multiome ATAC + Gene Expression v1 chemistry, and scRNA-seq libraries were generated using the Single Cell 3' v3 chemistry. Nuclei isolation for scMultiome was performed using a modified 0.1X lysis protocol from 10x Genomics protocol CG000366, with the digitonin concentration adjusted from 0.001% to 0.005%.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Mus musculus

SUBMITTER: Ridvan Cetin

PROVIDER: E-MTAB-16347 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Distinct roles of Atf3, Zfp711 and Bcl6b in early embryonic hematopoietic and endothelial lineage specification.

Cetin Ridvan R Picco Giulia G van Staalduinen Jente J Bindels Eric E Hoogenboezem Remco R van Beek Gregory G Sanders Mathijs A MA Fidan Yaren Y Korkmaz Ahmet A Gribnau Joost J van Haren Jeffrey J Huylebroeck Danny D Mulugeta Eskeatnaf E Grosveld Frank F

Development (Cambridge, England) 20251208 23

Hematopoiesis occurs in three consecutive overlapping waves in mammals, regulated by transcription factors. We investigated the role of three relatively poorly studied transcription factors in early embryonic hematopoietic development at single-cell resolution: Atf3, Zfp711 and Bcl6b. These transcription factors are upregulated early in development, when hematopoietic and endothelial lineages separate from cardiac and other mesodermal lineages. We combined multiplexed single-cell RNA sequencing ...[more]

PMID: 41200855

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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.

Project description:We aimed to identify the nature of iPSC and iPSC-neuro clones identified in E-MTAB-14065. We evaluated the combined effect on the epigenome and transcription at the single-cell level. This experiment demonstrated the importance of clonal tracking when performing scRNAseq screenings and allowed us to validate iPS2-multi-seq. Sample preparation for iPS2-multi-seq was performed following the 10X Genomics demonstrated protocol for nuclei isolation (CG000365, Rev D) and the 10X Multiome User Guide (CG000338, Rev F). Cells were detached using Versene and incubated with DNase I (200 U/mL) to reduce clumping, then washed and filtered through a 40 µm strainer (Flowmi). Cell viability and count were assessed using trypan blue staining (Sigma) on a hemocytometer. HiPSC cells P3 transfection pools were kept separately, and nuclei were pooled only prior to loading on the 10X Chromium. After cell lysis using freshly prepared buffer containing 0.1% Tween-20, IGEPAL CA-630, 0.01% digitonin, 1% BSA, and 1U/ml RNAse inhibitor, nuclei were isolated, washed twice with chilled wash buffer, and counted using both trypan blue and 1:1000 YOYO-1 staining (Invitrogen). Final nuclei counts were pooled 1:1 and diluted to 7,000 nuclei/μL before loading into the microfluidic chip. Nuclei were loaded after diluting in 10X Nuclei buffer according to the user guide. Chromatin tagmentation and reverse transcription were performed according to the 10X protocol. Libraries were indexed and amplified: 7 cycles for ATAC index PCR and 6 cycles for cDNA amplification. D5000 Tapestation and dsDNA hi-sensitivity Qubit analyses revealed appropriate fragment sizes and concentrations. The iPS2-seq library was obtained from the cDNA of the 10X gene expression library. Adaptations of the current protocol were necessary to obtain clean iPS2-seq libraries from the multiome cDNA library. Reduced cycle amplification to 11 cycles and increased input material to 200ng was key to this optimization, resulting in libraries with cleaner profiles, confirmed by D5000 Tapestation analysis and improved size distributions following 1.8x–0.7x SPRIselect purification. Libraries were further amplified and indexed using TT indices and purified again using a double-sided size selection.Sequencing was performed on an Illumina NextSeq 1000 using two flow cells P2 Xleap 100 cycles with paired-end dual indexing. ATAC libraries were sequenced at 400 million reads with 650 pM loading concentration and 1% PhiX spike-in (R1: 50 bp; i7: 8 bp; i5: 24 bp; R2: 49 bp). The gene expression library was pooled with the iPS2seq library with a 1:20 ratio, accounting for 380M reads for the gene expression and 20M reads for the iPS2seq library. Final pooled libraries were quantified using Qubit and loaded 650pM and 1% PhiX spike-in (R1: 28 bp; i7/i5: 10 bp; R2: 90 bp). iPS2-seq library generation required further optimization, compared to the original protocol described here, cDNA from 10X multiome as template for iPS2-seq PCR, required reduced cycle amplification and increased input material (up to 200 ng, 11 cycles), resulting in libraries with cleaner profiles, confirmed by D5000 Tapestation analysis and improved size distributions following 1.8x–0.7x SPRIselect purification.

Dataset Information

Single-Cell Multi-omics RNA and ATAC Sequencing of In Vitro Hematoendothelial Differentiation from Mouse Embryonic Stem Cells

Publications

Distinct roles of Atf3, Zfp711 and Bcl6b in early embryonic hematopoietic and endothelial lineage specification.

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