ABSTRACT: 10x linked-read sequencing data for individual HV31 generated using DNA from CD14+ monocytes, to a sequencing depth of ~40×. Sequencing was performed at Bart’s and the London Genome Centre on the Illumina HiSeq platform.
Project description:MGI single-tube long fragment read (stLFR) linked-read sequencing data for individual HV31 generated using DNA from CD14+ monocytes, to a sequencing depth of ~51×.
Project description:Oxford Nanopore long-read sequencing data for individual HV31 generated using DNA from CD14+ monocytes, to a sequencing depth of ~63×. Sequencing was performed at the Wellcome Centre for Human Genetics using the Oxford Nanopore PromethION platform.
Project description:PacBio continuous long read (CLR) sequencing data for individual HV31 generated on PacBio Sequel II instrument, using DNA from CD14+ monocytes, to a sequencing depth of ~35×. Sequencing was performed at the Wellcome Sanger Institute.
Project description:PacBio long-read circular consensus (CCS) sequencing data for individual HV31 generated on PacBio Sequel II instrument, using size-selected (10-15 kb) DNA from CD14+ monocytes, to a sequencing depth of ~12×. Sequencing was performed at the Wellcome Sanger Institute.
Project description:Monocytes are a heterogeneous cell population with subset-specific functions and phenotypes. The differential expression of CD14 and CD16 distinguishes classical CD14++CD16-, intermediate CD14++CD16+ and non-classical CD14+CD16++ monocytes. However, CD14++CD16+ monocytes remain the most poorly characterized subset so far. Therefore we analyzed the transcriptomes of the three monocyte subsets using SuperSAGE in combination with high-throughput sequencing. Analysis of 5,487,603 tags revealed unique identifiers of CD14++CD16+ monocytes, delineating these cells from the two other monocyte subsets. CD14++CD16+ monocytes were linked to antigen processing and presentation (e.g. CD74, HLA-DR, IFI30, CTSB), to inflammation and monocyte activation (e.g. TGFB1, AIF1, PTPN6), and to angiogenesis (e.g. TIE2, CD105). Therefore we provide genetic evidence for a distinct role of CD14++CD16+ monocytes in human immunity. Human monocyte subsets (CD14++CD16-, CD14++CD16+, CD14+CD16++) were isolated from 12 healthy volunteers based on MACS technology. Total RNA from monocyte subsets was isolated and same aliquots from each donor and monocyte subset were matched for SuperSAGE. Three SuperSAGE libraries (CD14++CD16-, CD14++CD16+ and CD14+CD16++) were generated.
Project description:Monocytes are a heterogeneous cell population with subset-specific functions and phenotypes. The differential expression of CD14 and CD16 distinguishes classical CD14++CD16-, intermediate CD14++CD16+ and non-classical CD14+CD16++ monocytes. However, CD14++CD16+ monocytes remain the most poorly characterized subset so far. Therefore we analyzed the transcriptomes of the three monocyte subsets using SuperSAGE in combination with high-throughput sequencing. Analysis of 5,487,603 tags revealed unique identifiers of CD14++CD16+ monocytes, delineating these cells from the two other monocyte subsets. CD14++CD16+ monocytes were linked to antigen processing and presentation (e.g. CD74, HLA-DR, IFI30, CTSB), to inflammation and monocyte activation (e.g. TGFB1, AIF1, PTPN6), and to angiogenesis (e.g. TIE2, CD105). Therefore we provide genetic evidence for a distinct role of CD14++CD16+ monocytes in human immunity.
Project description:Background: Monocytes play a crucial role in innate immune responses for host defense, yet their involvement in chronic obstructive pulmonary disease (COPD) remains poorly understood. We previously identified a subset of monocytes in COPD lung tissues characterized by high interleukin-6 receptor (IL-6R) expression. This study aimed to characterize the phenotypes of IL-6Rhi monocytes in the lung of COPD patients. Methods: Using flow cytometry, we assessed the abundance of pulmonary CD14+IL-6Rhi cells in never smokers (CNS), control ex-smokers (CES) and COPD patients. IL-6 expression in CD14+ monocytes from peripheral blood of patients with COPD was also examined. CD45+CD206–CD14+IL-6Rhi and CD45+CD206–CD14+IL-6R– cells were isolated from COPD lung tissues for transcriptome analysis. A monocyte line THP1 cell with constitutive IL-6R expression was stimulated with recombinant IL-6, followed by RNA sequencing to evaluate IL-6 responsiveness of IL-6R+ monocytes. Results: The number of pulmonary CD14+IL-6Rhi monocytes was elevated in COPD patients compared to CNS, while CD14+ monocytes in the peripheral blood of COPD patients did not express IL-6R. Upregulated mRNA expression in CD14+IL-6Rhi monocytes was associated with chemotaxis, monocyte differentiation, fatty acid metabolism and integrin-mediated signaling pathway. Stimulation of THP1 cells with recombinant IL-6 induced gene expression changes linked to chemotaxis and organism development. Conclusion: In patients with COPD, CD14+IL-6Rhi monocytes increase in lung tissues compared to CNS and exhibit a different transcriptome profile from CD14+IL-6R– monocytes.
Project description:Background: Monocytes play a crucial role in innate immune responses for host defense, yet their involvement in chronic obstructive pulmonary disease (COPD) remains poorly understood. We previously identified a subset of monocytes in COPD lung tissues characterized by high interleukin-6 receptor (IL-6R) expression. This study aimed to characterize the phenotypes of IL-6Rhi monocytes in the lung of COPD patients. Methods: Using flow cytometry, we assessed the abundance of pulmonary CD14+IL-6Rhi cells in never smokers (CNS), control ex-smokers (CES) and COPD patients. IL-6 expression in CD14+ monocytes from peripheral blood of patients with COPD was also examined. CD45+CD206–CD14+IL-6Rhi and CD45+CD206–CD14+IL-6R– cells were isolated from COPD lung tissues for transcriptome analysis. A monocyte line THP1 cell with constitutive IL-6R expression was stimulated with recombinant IL-6, followed by RNA sequencing to evaluate IL-6 responsiveness of IL-6R+ monocytes. Results: The number of pulmonary CD14+IL-6Rhi monocytes was elevated in COPD patients compared to CNS, while CD14+ monocytes in the peripheral blood of COPD patients did not express IL-6R. Upregulated mRNA expression in CD14+IL-6Rhi monocytes was associated with chemotaxis, monocyte differentiation, fatty acid metabolism and integrin-mediated signaling pathway. Stimulation of THP1 cells with recombinant IL-6 induced gene expression changes linked to chemotaxis and organism development. Conclusion: In patients with COPD, CD14+IL-6Rhi monocytes increase in lung tissues compared to CNS and exhibit a different transcriptome profile from CD14+IL-6R– monocytes.
Project description:The goal of this experiment was to explore how human platelets affect the transcriptional responses of primary human CD14+ blood monocytes to lipopolysaccharide (LPS), and NLRP3 activation with Nigericin. For this purpose, we analyzed the mRNA expression of 770 myeloid-specific transcripts using the nCounter® Nanostring Human Myeloid Innate Immunity Panel v2. We isolated classical monocytes (CD14+CD16−) from the peripheral blood of healthy volunteers. Monocytes were derived from PBMCs using negative selection with the EasySep™ Human Monocyte Isolation Kit from STEMCELLTM Technologies. We modified this process by either including or excluding a platelet-depleting cocktail, creating two groups: \\"Standard Monocytes (StdMo)\\" and \\"Platelet-depleted Monocytes (PdMo).\\" To examine the impact of platelets further, we supplemented PdMos with fresh autologous platelets at a ratio of 50 platelets per monocyte, resulting in a third group, \\"PdMo + Plts.\\" StdMos were prepared according to the standard protocol provided by the EasySep™ Kit. For the PdMos, a Platelet Removal Component (50 µl ml−1) from the kit was used during isolation. We also reconstituted platelet-depleted monocytes with platelets and analyzed platelets alone to determine their specific mRNA contributions. The groups—StdMo, PdMo, PdMo + Plts, and platelets alone—were then exposed to 2 ng/ml of Ultrapure LPS (from E. coli O111:B4) for 4.5 hours, or stimulated with LPS for 3 hours followed by inflammasome activation with Nigericin (10 µM) for 90 min before extracting RNA for analysis.
Project description:Comparison of the DNA methylation profiles of CD14+ monocytes from human peripheral blood with derived dendritic cells (DCs) and macrophages (MACs) obtained by exposure with GM-CSF/IL-4 and GM-CSF, respectively. Effects on the methylation profiles of DCs and MACs of JAK3 inhibitor PF-956980 The methylation profiles of bisulfite-modified DNA of human CD14+ monocytes were compared with derived dendritic cells (DCs), macrophages (MACs) following GM-CSF/IL-4 and GM-CSF incubation, and DC and MAC samples incubated with JAK3 inhibitor PF-956980 using the Infinium HumanMethylation450 BeadChips (Illumina, Inc., San Diego, CA,). This platform allows the interrogation of >485,000 methylation sites per sample at single-nucleotide resolution, and comprises an average of 17 CpG sites per gene in the 99% of RefSeq genes. 96% of CpG islands are covered, with additional coverage in CpG island shores and the regions flanking them. The samples were hybridized in the array following the manufacturerâÂÂs instructions. Total DNA isolated by standard procedures from CD14+ cells (total monocytes, MOs) corresponding to three sets of samples of monocytes (MOs), derived DCs and MACs (DCs and iMACs; DMSO as these samples were differentiated in the absence of JAK3 inhibitors) and DCs and MACs differentiated in the presence of JAK3 inhibitor PF-956980.