Project description:To detect the role of OP9 stromal cells in our optimized 3D self-assembling peptide induction system followed by the OP9 coculture system. First, we used RNA-seq to analyze mouse pluripotent stem cells derived total cells at day5 between the 3D+OP9 and 3D+0.1% group in our hematopoietic differentiation system. In addition, to further evaluate hematopoietic transcriptome differences between Lin-Sca-1+c-kit+CD201+ cells and Lin-Sca-1+c-kit+CD201- cells, we used RNA-seq to analyze mouse pluripotent stem cells derived Lin-Sca-1+c-kit+CD201+ and Lin-Sca-1+c-kit+CD201- cells at day5 in our 3D+OP9 hematopoietic differentiation system.Meanwhile,we used the mouse embryonic stem cell(mESC) as negative control and bone marrow derived Lin-Sca-1+c-kit+CD201+ and fetal liver derived Lin-Sca-1+c-kit+CD201+ as positive control.
Project description:RNA sequenicg was performed on mouse bone marrow GMPs (Lin-, cKit+, Sca1-, CD34+, CD16/32 hi) labeled by osteoblastic cell derived extracellular vesicles as indicated by GFP labeling. Cells were isolated by FACS sorting from the bone marrow of Ocn-GFPTopaz mice and total RNA was extracted using the miRNeasy micro kit from (Qiagen)
Project description:Small RNA sequenicg was performed on mouse bone marrow GMPs (Lin-, cKit+, Sca1-, CD34+, CD16/32 hi) labeled by osteoblastic cell derived extracellular vesicles as indicated by GFP labeling. Cells were isolated by FACS sorting from the bone marrow of Ocn-GFPTopaz mice and total RNA was extracted using the miRNeasy micro kit from (Qiagen)
Project description:We discovered that mice that lack Lsd1 in hematopoietic cells were exhibited increased frequencies of CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs, but completely lacked the lin- c-Kit+ Sca-1- myeloid progenitor compartment. To determine the genes altered by Lsd1-loss, CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs from Lsd1fl/fl and Lsd1fl/fl Mx1Cre mice were FACS-purified to be analyzed by gene expression profiling. Primary CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs were isolated by FACS-sorting, from the bone marrow of Lsd1fl/fl and Lsd1fl/fl Mx1Cre animals, one week after the final p(I:C) dose. Total RNA from three biological replicates per genotype was extracted with the RNeasy Micro Kit (QIAGEN), treated with DNaseI, reverse transcribed. RNA extracted from CD150+ CD48- lin- c-Kit+ Sca-1+ cells was amplified with the Ovation Pico WTA RNA Amplification System 2 (NuGEN Technologies). Single-stranded cDNA amplification products were purified using QIAquick PCR Purification Kit (QIAGEN) and labeled with the FL-Ovation cDNA Biotin Module V2 (NuGEN). The Biotin-labeled cDNA was used to hybridize to Affymetrix expression arrays using the Mouse Genome 430 2.0 array platform. Primary CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs were isolated by FACS-sorting, from the bone marrow of Lsd1fl/fl and Lsd1fl/fl Mx1Cre animals, one week after the final p(I:C) dose. Total RNA from three biological replicates per genotype was extracted with the RNeasy Micro Kit (QIAGEN), treated with DNaseI, reverse transcribed. RNA extracted from CD150+ CD48- lin- c-Kit+ Sca-1+ cells was amplified with the Ovation Pico WTA RNA Amplification System 2 (NuGEN Technologies). Single-stranded cDNA amplification products were purified using QIAquick PCR Purification Kit (QIAGEN) and labeled with the FL-Ovation cDNA Biotin Module V2 (NuGEN). The Biotin-labeled cDNA was used to hybridize to Affymetrix expression arrays using the Mouse Genome 430 2.0 array platform. Primary CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs were isolated by FACS-sorting, from the bone marrow of Lsd1fl/fl and Lsd1fl/fl Mx1Cre animals, one week after the final p(I:C) dose. Total RNA from three biological replicates per genotype was extracted with the RNeasy Micro Kit (QIAGEN), treated with DNaseI, reverse transcribed. RNA extracted from CD150+ CD48- lin- c-Kit+ Sca-1+ cells was amplified with the Ovation Pico WTA RNA Amplification System 2 (NuGEN Technologies). Single-stranded cDNA amplification products were purified using QIAquick PCR Purification Kit (QIAGEN) and labeled with the FL-Ovation cDNA Biotin Module V2 (NuGEN). The Biotin-labeled cDNA was used to hybridize to Affymetrix expression arrays using the Mouse Genome 430 2.0 array platform.
Project description:compare the gene expression profile between irradiated Lin-Sca-1+c-Kit+ (LSK) cells from mouse bone marrow reconstituted with wild type and necdin null fetal liver cells The Affymetrix oligonucleotide array was used for this analysis compare the gene expression profile betweenirradiated Lin-Sca-1+c-Kit+ (LSK) cells from mouse bone marrow reconstituted with wild type and necdin null fetal liver cells
Project description:compare the gene expression profile between irradiated Lin-Sca-1+c-Kit+ (LSK) cells from mouse bone marrow reconstituted with wild type and necdin null fetal liver cells The Affymetrix oligonucleotide array was used for this analysis
Project description:Bulk RNA-seq was performed on murine Lin-Sca+Kit+ bone marrow cells from a transgenic SCLtTA/BCR-ABL (DTG) mouse BM cells (CD45.2) to understand the molecular consequences of BCR-ABL1 expression on the global mRNA programme of these cells.
Project description:The study was a comparison of gene expression using RNA-seq. We analyzed the stem and progenitor cells from WT and Vav-cre+ Tet2fl/fl Flt3-ITD (T2F3) mice. We isolated stem cells LSK (lin- sca+ kit+) and granulocyte-macrophage progenitors GMP (lin- sca- kit+ fcgr+ cd34+) cells from bone marrow. Comparisons were made across genotypes WT vs. T2F3 and cell types LSK vs. GMP.
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.
