Project description:Here, we present a Small RNA-Seq dataset of isolated mouse hematopoietic stem cells (HSC LSK slam; Lineage- Sca-1+ c-Kit+ CD150+CD48-) of Meg3 KO (induced MxCre Meg3mat flox/pat wt) and control (induced MxCre) cells
Project description:LRF, which is encoded by the ZBTB7A gene and formerly known as POKEMON (POK erythroid myeloid ontogenic factor), was originally identified as a PLZF (promyelocytic leukemia zinc finger) homologue interacting with BCL6 (B-cell lymphoma 6). LRF is a transcription factor that is broadly expressed in hematopoietic lineage cells, but its expression is particularly high in erythroblasts and germinal center (GC) B-cells. The goal of this study is to assess the effect of LRF loss on the LT-HSC transcriptome. Nine days after injection of adult mice with polyinosinic polycytidylic acid (pIpc) to activate Cre, total RNAs were isolated from double-sorted LT-HSCs from LRF Flox/+ Mx1-Cre+ and LRF Flox/Flox Mx1-Cre+ mice and processed for microarray analysis. We performed gene expression microarray analysis of FACS-sorted LT-HSCs (LSK IL7Ra-Flt3-CD150+CD48-) to assess the effect of Lrf loss on the LT-HSC transcriptome. Zbtb7a Flox/+ Mx1-Cre+ mice were used as a control to normalize the potential effects of Cre recombinase. LT-HSCs were FACS-sorted from three Lrf knockout (Zbtb7a Flox/Flox Mx1-Cre+) and two control (Zbtb7a Flox/+ Mx1-Cre+) mice, nine days after the first pIpC injection.
Project description:In this experiment we sought to identify region with differential PRC2, JARID2, and H3K27me3 occupancy in human induced pluripotent stem cell lines with silenced or expressed MEG3 locus to indirectly determine the effect of this ncRNA on PRC2 function. 8 hIPSC lines, 5 MEG3+ and 3 MEG3-; ChIP-seq for EZH2, JARID2, and H3K27me3. Some done in replicates.
Project description:To begin to explore mechanisms by which microbiota signals regulate HSC lineage bias, gene expression profiling was performed on sorted LSK-SLAM cells from aged SPF and aged GF mice.
Project description:In this study, we use a conditional mouse model for Cebpa to investigate the significance of C/EBPα in HSCs. The frequency of HSCs is unaltered following deletion of C/EBPα, however, upon serial transplantations of either full BM or purified HSCs, the stem cells and stem cell activity is lost. This is not due to increased proliferation, but rather caused by a shift from quiescence to apoptosis with a resultant exhaustion of the stem cell pool. We identify direct C/EBPα target genes by combining genome-wide C/EBPα ChIP-seq analysis in stem and progenitor cells with gene expression data from HSC with and without C/EBPα. Furthermore, we explore the impact of C/EBPα on active and repressive histone modifications by doing functional genome-wide ChIP-seq analysis of H3K4Me3 and H3K27Me3 in stem and progenitor cells with and without C/EBPα. We have sorted HSCs from 3 Cebpaflox/flox and 4 Cebpaflox/flox;Mx1Cre mice 18 days after pIC injection.
Project description:Bone marrow Hdc-GFPhi and Hdc-GFPlo HSPC (SLAM-LSK, Lin-c-kit+Sca-1+CD150+CD48-) HSCs were isolated from mouse femur and tibia from histidine decarboxylase (Hdc) green fluorescent protein (Hdc-GFP) mice. Hdc-GFPhi HSC and Hdc-GFPlo HSC cells were sorted by combinations of GFP and the cell surface markers of HSC. total RNA was isolated from sorted 2,500 HSPCs using ARCTURUS PicoPure RNA isolation kit (Life Technologies). cDNA was amplified and libraries were constructed by using SMARTer Ultra Low Input RNA kit (Clontech Laboratories) and Nextera XT DNA Library Preparation kit (Illumina) according to manufacturer’s instructions respectively. Sequencing was performed on the Illumina HiSeq2000 platform.
Project description:To determine genes in FL HSCs that are sensitive to Notch signagling, E14.5 FL cells were cultured on DL1( to stimulate Notch signaling). Cells were cultured in the presence of DMSO (vehicle control) or gamma secretase inhibitor (1uM) for 4 hrs or 10hrs. Gamma secretase inhibitor was used to inhibit Notch signaling. SLAM-LSKs were sorted and used for RNA preparation. 7 biological replicates (14 totalt samples) were collected for cells cultured for 4hrs. 4 biological replicates (8 total samples) were collected from cells cultured for 10hrs. Each replicate consisted of a DMSO and GSI treated sample
Project description:In this study we demonstrate that Tgif1 has a role in HSCs maintenance, self-renewal and quiescence. RNA sequencing data of LSK cells (HSCs enriched cell population) from Tgif1-/- and wild type mice implicates that multiple pathways involved in HSC quiescence and self-renewal are disturbed in Tgif1 deficient mice. RNA expression profiles of wild type (WT) and Tgif1-/- LSK cells were generated by RNA sequencing, in triplicate, using Illumina HiSeq 2000.
Project description:Ezh2 is a protein member of PRC2 and has described like a functional repressor gene. We are investigating the role of Ezh2 in hematopoietic stem cell, aging and leukemia. In the present study, we generated mouse models that allow gain-of-function of Ezh2 in the hematopoietic system. Activation of Ezh2 expression specifically in self-renewing HSCs alters gene expression programs and severely compromises hematopoietic function, leading to the development of myeloproliferative disease. We used microarrays to detail the global programme of gene expression in LSK that has relationship with the overexpression of Ezh2. LSK were marked and isolated by Fluorescence-activated cell sorting in Trizol.
Project description:The adaptor protein MERIT40 is a core subunit of deubiquitinating (DUB) complexes that specifically cleave Lysine63-polyubiquitin chains. We found that MERIT40 is an important negative regulator of hematopoietic stem cell (HSC) homeostasis, quiescence and self-renewal. This study aims to investigate the molecular mechanism by which MERIT40 regulates HSC expansion and cell cycle. We performed expression profiling of bone marrow CD150+CD48-LSK LT-HSCs from WT and Merit40-/- mice. Results identify select MERIT40-mediated pathways with potential involvement in HSC cell cycle regulation. CD150+CD48-LSK HSCs were double sorted from WT and Merit40-/- mice at young age. RNA was isolated using miRNeasy kit from QIAGEN and processed using the NuGEN Pico kit. The microarray analysis was performed at the PENN Molecular Profiling/Genomics Facility using GeneChip Mouse Gene 2.0ST array (Affymetrix).