Single-cell RNA sequencing of RUNX1-RUNX1T1(9a) transformed mouse bone marrow cells
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ABSTRACT: Single-cell RNA sequencing analysis of RUNX1-RUNX1T1(9a) transformed c-kit positive cells with (Kat2a WT) and without Kat2a (Kat2a NULL). Lineage negative bone marrow cells were collected from Kat2a fl/fl Mx1-Cre-/- and Kat2a fl/fl Mx1-Cre +/- animals after pIpC treatment and transduced with RUNX1-RUNX1T1(9a) expressing retrovirus (reported by GFP expression). Cells were injected into irradiated C57BL6 mice and GFP positive c-Kit positive bone marrow cells collected 2 and 4 months after transplantation. Cells were processed for single-cell RNA sequencing library preparation (10X chromium single cell) and next gene sequencing following 10X genomics v2 protocol.
Project description:Epcam-positive cells from fetal intestinal epithelium were FACS sorted and processed using 10X genomics to characterise the differentiation of fetal intestinal cells at E16.5.
Project description:To clarify the role of AML1-ETO in aberrant hematopoiesis, we generated conditional AML1-ETO knock-in mice. Our research indicated AML1-ETO induction impaired bone marrow hematopoietic reconstitution and resulted in aberrant accumulation of Lin-Sca-1+c-Kit+ (LSK) cells. To clarify the potential mechanism, we compared gene expression profiling and epigenetic alterations of LSK cells between AML1/ETO mouse (conditional knock-in of Runx1-Runx1t1; Mx1-Cre mouse model) and the control mouse (AML1/ETO mice without Mx1-Cre).
Project description:To clarify the role of AML1-ETO in aberrant hematopoiesis, we generated conditional AML1-ETO knock-in mice. Our research indicated AML1-ETO induction impaired bone marrow hematopoietic reconstitution and resulted in aberrant accumulation of Lin-Sca-1+c-Kit+ (LSK) cells. To clarify the potential mechanism, we compared gene expression profiling and epigenetic alterations of LSK cells between AML1/ETO mouse (conditional knock-in of Runx1-Runx1t1; Mx1-Cre mouse model) and the control mouse (AML1/ETO mice without Mx1-Cre).
Project description:To clarify the role of AML1-ETO in aberrant hematopoiesis, we generated conditional AML1-ETO knock-in mice. Our research indicated AML1-ETO induction impaired bone marrow hematopoietic reconstitution and resulted in aberrant accumulation of Lin-Sca-1+c-Kit+ (LSK) cells. To clarify the potential mechanism, we compared gene expression profiling and epigenetic alterations of LSK cells between AML1/ETO mouse (conditional knock-in of Runx1-Runx1t1; Mx1-Cre mouse model) and the control mouse (AML1/ETO mice without Mx1-Cre).
Project description:Comparison of the germinal center B cell receptor repertoire in Cxcl13-Cre/TdTom EYFP (control) and Cxcl13-Cre/TdTom EYFP Cxcl12fl/fl mice on day following subcutaneous immunization with NP-KLH.
Project description:FoxM1, a mammalian Forkhead Box M1 protein, is known as a typical proliferation-associated transcription factor that regulates of G1/S and G2/M transition in the proliferating cells. However, the in vivo function of FoxM1 in adult stem cells remains unknown. Here, we found that FoxM1 is highly expressed in hematopoietic stem cells (HSCs) and is essential for maintaining quiescence and self-renewal of HSCs in vivo. FoxM1-deficient mice developed leukopenia, thrombocytopenia and neutropenia with an approximately 6-fold decrease in HSC pool size, which is associated with a failure of G0 cell cycle regulation and increased cell cycling in HSCs. FoxM1 absence did not affect lineage commitment of HSCs and progenitors. However, FoxM1 loss significantly reduced the repopulating capacity and self-renewal of long-term HSC in a cell-autonomous manner. Mechanistically, FoxM1 loss markedly down-regulates the expression of orphan nuclear receptor Nurr1, known to regulate HSC quiescence. We found that FoxM1 directly bound the promoter region of Nurr1 and induced transcriptional activity of Nurr1 promoter in vitro, and forced expression of Nurr1 rescued FoxM1-deletion-induced G0 loss of HSC-enriched population in vitro. Thus, our studies show a previously unrecognized role of FoxM1 as a critical regulator of HSC quiescence and self-renewal by controlling Nurr1-mediated pathways. The Hematopoietic Stem Cells (HSCs) were sorted from FoxM1[fl/fl] and Tie2-Cre FoxM1[fl/fl] mice, then amplified with Ovation Pico WTA System V2 before microarray analysis. There are 3 samples from FoxM1[fl/fl]mice and 3 samples from Tie2-Cre FoxM1[fl/fl] mice.
Project description:Live CD45+ cells from the peripheral blood (PB) and synovial tissue (ST) of 3 naive to treatment RA patients were sorted for single cell RNA sequencing (scRNAseq) using 10x Genomics platform. Library preparation was performed using 10X Single Cell 3’ Reagent Kits v2 and libraries were sequenced on the Illumina HiSeq 4000 system to a minimum depth of 50k reads/cell.
Project description:We used the resolving power of single-cell transcriptional profiling to molecularly characterize the mouse adipose stem and progenitor cell-enriched, subcutaneous adipose stromal vascular fraction. We molecularly assessed CD45- CD31- SVF cells using the 10x Genomics Chromium (10x) platform.
Project description:The chemokines CXCL13 and CXCL12 are reported to be important for the germinal center reaction. Since CXCL12-deficient mice are embryonically lethal, here we took advantage of the Cxcl13-Cre/TdTomato mouse models to genetically ablate CXCL12 from B cell-interacting reticular cells and examine the molecular consequence on germinal center B cells. Spatial segregation of follicular dendritic cells, germinal center B cells and follicular helper T cells is impaired in Cxcl13-Cre/TdTomato Cxcl12fl/fl mice. Single cell transcriptomic analysis revealed that all germinal center B cell subsets (corresponding to distinct stages of the germinal center response) are present in draining lymph nodes of immunized CXCL12-conditionally deficient mice. While most transcriptional regulators of the germinal center response are unperturbed by the genetic perturbation of CXCL12, Bach2 levels were elevated in germinal center B cells from lymph nodes of Cxcl12fl/fl mice. Moreover, single cell B cell receptor sequencing revealed that germinal center B cells in Cxcl13-Cre/TdTomato Cxcl12fl/fl mice harbour a lower mutational burden when compared to germinal center B cells isolated from immunized control mice. Gene expression profiles were validated by flow cytometry and suggest that the provision of CXCL12 by reticular cells governs efficient germinal center responses.
Project description:To understand kinase-dependent and kinase-independent functions of Cdk6 in the hematopoietic stem and progenitor compartment, mouse models carrying wild-type Cdk6 (Cdk6_WT), a homozygous knock-in of a kinase-inactivated variant of Cdk6 (Cdk6_K43M) or a homozygous Cdk6 knock-out (Cdk6_KO) were used. From these mice, lineage-negative, Sca1-positive, c-Kit-positive cells (LSK cells) were isolated by means of flow cytometry cell sorting. 30000 LSK cells per mouse were subjected to library prep for single cell RNA Sequencing using the Chromium NextGem Single Cell 5’ v2 Kit (10x Genomics, Pleasanton, CA, USA)