Transcriptional program of terminal granulocytic differentation
Ontology highlight
ABSTRACT: To characterize the transcriptional program that governs terminal granulocytic differentation in vivo, we performed comprehensive microarray analysis of human bone marrow population highly enriched for promyelocytes, myelocytes / metamyelocytes and neotrophils. Bone marrow and peripheral blood samples were collected from healthy individuals in parallel. Cell populations representing successive stages of terminal granulocytic differentation were isolated from human bone marrow samples by 2-layer density gradient centrifugation, neutrophils were collected from peripheral blood using 1-layer density gradient centrifugation. All populations were depleted of nongranulocytic cells by immunomagnetic sorting.
Project description:To characterize the transcriptional program that governs terminal granulocytic differentation in vivo, we performed comprehensive microarray analysis of human bone marrow population highly enriched for promyelocytes, myelocytes / metamyelocytes and neotrophils.
Project description:How ferritin regulates neutrophil leukocyte function remains unknown. To address this question, we injected mice with ferritin. bone marrow-derived neutrophils (BMDN) were isolated by density gradient centrifugation. Then we displayed RNA-seq of isolated BMDNs.
Project description:Purpose: The goal of this study is to investigate the role of integrin beta3 (Itgb3) deficiency on the gene expression of bone marrow cells. Methods: Bone marrow was harvested by flushing the femurs and tibias of Apoe(-/-), Itgb3(+/+) and Apoe(-/-), Itgb3(-/-) mice in PBS with 2% fetal bovine serum on ice. Cells were isolated from bone marrow by density gradient centrifugation using Lympholyte (Cedarlane, CL0531). After centrifugation at 1300 g for 20 min, bone marrow cells were collected from the interface and washed in Ca2+/Mg2+ - free HBSS and dissociated to a single-cell suspension by filtering through a 70-µm nylon mesh. The cells were then subjected to 10X Genomics single cell 3’ RNA-seq libraries construction and sequencing. scRNA-seq data was processed with Cell Ranger v 3.1.0. Reads were aligned to a modified version of the mouse transcriptome mm10. The top cell barcodes selected by Cell Ranger were utilized for downstream analysis. Results: Expression profiles of the transcriptome are compared between cell types isolated from the bone marrow of Apoe(-/-), Itgb3(+/+) and Apoe(-/-), Itgb3(-/-) mice. Conclusions: Significant differences in the expression profile of genes are present in bone marrow cells of Apoe(-/-), Itgb3(+/+) and Apoe(-/-), Itgb3(-/-) genotypes.
Project description:Purpose: The goal of this study is to investigate the gene expression in aged (18 month old) and young (3 month old) bone marrow-derived monocytes. Methods: Bone marrow was harvested by flushing the femurs and tibias of young (3 month old) and aged (18 month old) mice in PBS with 2% fetal bovine serum (FBS) on ice. Cells were isolated from bone marrow by density gradient centrifugation using Lympholyte (Cedarlane, CL0531). After centrifugation at 1300 g for 20 min, bone marrow cells were collected from the interface and washed in Ca2+/Mg2+ - free HBSS. The cells were dissociated to a single-cell suspension by filtering through a 70-µm nylon mesh. For monocyte isolation, a suspension of bone marrow cells was stained with Alexa 700 anti-CD3 (1:100, BioLegend, 100215), PE-Cy7 anti-CD19 (1:100, BioLegend, 115520), FITC anti-CD11b (1:100, BD Pharmingen, 553310) and APC anti-Ly6c antibodies (1:100, BioLegend, 128015) in PBS with 0.5% FBS for 30 min. CD11b+Ly6c+CD3-CD19- monocytes were isolated with a BD FACSAria cell sorter. Total RNA was extracted and subjected to bulk RNA-seq using Illumina HiSeq 2000. Results: Expression profiles of the transcriptome are compared between bone marrow-derived monocytes isolated from aged (18 month old) and young (3 month old) mice. Conclusions: Significant differences in the expression levels of genes are noted between the two age groups.
Project description:Bone marrow aspirates were obtained from patients with relapsed/refractory large B cell lymphoma (rrLBCL), mononuclear cells isolated by ficoll density-gradient centrifugation, and loaded onto a 10X Chromium for single cell RNA-sequencing using 5’ chemistry without prior cryopreservation. Healthy donor bone marrow mononuclear cells were obtained from healthy allogeneic stem cell transplant donors and analyzed following viable cryopreservation.
Project description:Bone marrow aspirates were obtained from patients with relapsed/refractory large B cell lymphoma (rrLBCL), mononuclear cells isolated by ficoll density-gradient centrifugation, and loaded onto a 10X Chromium for single cell RNA-sequencing using 5’ chemistry without prior cryopreservation. Healthy donor bone marrow mononuclear cells were obtained from healthy allogeneic stem cell transplant donors and analyzed following viable cryopreservation.
Project description:The aim of this research was to explore activation/deactivation of signaling pathways in mononuclear cells from pediatric patients with acute myeloid leucosis and acute lymphoid leucosis. Mononuclear cells were separated shortly after bone marrow samples collection. Cells were obtained by a density gradient centrifugation method using Ficoll. Cells were dissolved in RNALater solution, aliquoted and stored at -20°C till RNA extraction and microarray hybridisation.
Project description:The aim of this research was to explore activation/deactivation of signaling pathways in mononuclear cells from pediatric patients with acute myeloid leucosis and acute lymphoid leucosis. Mononuclear cells were separated shortly after bone marrow or blood samples collection. Cells were obtained by a density gradient centrifugation method using Ficoll. Cells were dissolved in RNALater solution, aliquoted and stored at -20°C till RNA extraction and microarray hybridisation.
Project description:Genome wide DNA methylation profiling of 2 distinct subpopulations of sperm within a single ejaculate. The Illumina Infinium 450k Human DNA methylation Beadchip was used to obtain DNA methylation profiles across approximately 450,000 CpGs. Samples were provided by 20 normozoospermic individuals. A density gradient centrifugation was performed on each samples to yeild two distict populations for each ejaculate (one from the 90% isolate layer high quality sperm and one from the 35% isolate layer low quality sperm of the column).