Immune and HCT116 RNA mixtures
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Etienne Becht
PROVIDER: E-GEOD-64385 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:These experiments were designed as a benchmark tool for deconvolution methods. 5 immune cell populations were sorted from 3 healthy donors' peripheral bloods. Peripheral Blood Mononuclear Cells (PBCMs) and PolymorphoNuclear Cells (PMN) were separated using gradient centrifugation. T cells (DAPI-/CD3+/CD14-/CD19-/CD56-), monocytes (DAPI-/CD3-/CD14+/CD19-/CD56-), B cells (DAPI-/CD3-/CD14-/CD19+/CD56-) and NK cells (DAPI-/CD3-/CD14-/CD19-/CD56+) were FACS-sorted from PBMCs and neutrophils (DAPI-/CD66b+/CD19-/CD3-/CD56-/CD14-) were sorted from PMNs. RNA was extracted from the purified cell population, as well as from the HCT116 colon cancer cell line. RNAs from pure populations were then mixed in various proportions.
2016-02-18 | GSE64385 | GEO
Project description:we examined the effects of IL6 inhibition treatment on biologic molecular pathways in a 59 year old patient well diagnosed with RA with inadequate response to DMARD thus treated with tocilizumab. To assess the immunological outcome we used quantitative proteome analysis to evaluate changes in the proteome profile of different biologic pathways in leukocyte cell populations important to RA pathogenesis isolated by positive enrichment of CD14, CD4, CD8, CD19, and CD56.
2016-01-05 | PXD002686 | Pride
Project description:CD4+ T cells play a fundamental role in host defenses against viruses by orchestrating B cell development and/or by directly targeting pathogens. This dataset aimed at understanding the molecular profile of CD4+ T cells in the context of an acute and severe SARS-CoV-2 infection. The analyzed patient was recruited at Henri Mondor University Hospital (AP-HP, Paris France) in March 2020, and required oxygen as treatment. Peripheral (CD14-CD56-CD123-CD19-CD3+CD4+CD45RA-PD-1high) T cells were FACS-sorted using a Sony MA900 in PBS/0.08% FCS. 1.105 cells were obtained and 20000 were loaded in the 10x Chromium Controller to generate single-cell gel-beads in emulsion. The scRNA-seq libraries were generated using the Chromium Next GEM Single Cell V(D)J Reagent Kit v.1.1 with Feature Barcoding (10x Genomics) according to the manufacturer’s protocol. PBMCs were initially isolated from venous blood samples via standard density gradient centrifugation and used after cryopreservation at -150°C. Cells were thawed using RPMI-1640 (Gibco) 10% FBS, washed twice and incubated with fluorochrome-conjugated antibody cocktail and viable cells were identified using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) incubated with conjugated antibodies used for cell sorting (CD3/CD4/CD14/CD19/CD45/CD56/CD123/PD-1) as well as a panel of barcoded TotalSeqC® antibodies (CD11c/CD21/CD27/CD38/CD49d/CD69/CD71/CD73/CD95/CD103/CD138/CD305/CD307d/CD307e/CXCR3/CXCR4/CXCR5/HLA-DR/ICOS/ITB7/LAG-3/NKG2D/TIM3). A single sort was performed for this patient and 5’-transcriptomic, VDJ and ADT libraries were sequenced .
2023-09-30 | E-MTAB-13321 | biostudies-arrayexpress
Project description:Objective: Production of pathogenic autoantibodies by self-reactive plasma cells (PC) is a hallmark of autoimmune diseases. Investigating the prevalence of PC in autoimmune disease and their relationship with known pathogenic pathways may increase our understanding of the role of PC in disease progression and treatment response. Methods: We developed a sensitive gene expression based method to overcome the challenges of measuring PC using flow cytometry. Whole genome microarray analysis of sorted cellular fractions identified a panel of genes, IGHA, IGJ, IGKC, IGKV, and TNFRSF17, expressed predominantly in PC. The sensitivity of the PC signature score created from the combined expression levels of these genes was assessed through ex vivo experiments with sorted cells. This PC gene expression signature was used for monitoring changes in PC levels following anti-CD19 therapy; evaluating the relationship between PC and other autoimmune disease-related genes; and estimating PC levels in affected blood and tissue from multiple autoimmune diseases. Results: The PC signature was highly sensitive and capable of detecting as few as 300 PCs. The PC signature was reduced over 90% in scleroderma patients following anti-CD19 treatment and this reduction was highly correlated (r = 0.77) with inhibition of collagen gene expression. Evaluation of multiple autoimmune diseases revealed 30-35% of lupus, rheumatoid arthritis, and scleroderma patients with increased PC levels. Conclusion: This newly developed PC signature provides a robust and accurate method to measure PC levels in the clinic. Our results highlight subsets of patients across multiple autoimmune diseases that may benefit from PC depleting therapy. To examine gene expression in purified cellular fractions, normal human blood was collected from 2 donors as per institutional policy. The granulocyte (CD15+), monocyte (CD14+), T cell (CD3+), B cell (Non-PC gated, CD19+), and PC (CD27++CD38++) fractions from peripheral blood were separated. White blood cells were washed with FACS buffer (PBS + 0.5%BSA + 2mM EDTA (Gibco)) and incubated with 20% heat-inactivated FBS for 10-15 minutes on ice. The following mAbs were added directly to the cells: CD15 (HI98); CD14 (M5E2), CD3 (UCHT1), CD27 (M-T271), CD38 (HB7), and DAPI (Molecular probes). Cells were sorted on a Becton Dickinson FACS Aria II flow cytometer. All sorted fractions were collected in FACS buffer, centrifuged, and the resulting cell pellet was suspended in RNA lysis buffer (Ambion).
2013-03-28 | E-GEOD-45535 | biostudies-arrayexpress
Project description:Group 3 innate lymphoid cells (ILC3) are defined by the expression of RORM-NM-3t, which is selectively required for their development. The lineage-specified progenitor cells of human ILC3 and their developmental site after birth remain undefined. Here we identified a novel population of human CD34+ hematopoietic progenitor cells (HPC) expressing RORM-NM-3t and sharing with ILC3 a distinct transcriptional signature. RORM-NM-3t+ CD34+ HPC were located in tonsils and intestinal lamina propria (LP) and selectively differentiated towards ILC3. Conversely, RORM-NM-3t- CD34+ HPC displayed commitment potential for both ILC3 and NK cells and the differentiation fate towards these two cell lineages was determined by cytokine and aryl hydrocarbon receptor (AhR) signaling. Thus, we propose that RORM-NM-3t+ CD34+ cells represent human lineage-specified progenitors of IL-22+ ILC3 and that tonsils as well as intestinal LP might be preferential sites of their differentiation. ILC3, NK cells and the CD34+ HPC subsets were sorted from tonsils of 6 distinct donors to purity above 95%. cRNA of the sorted cell populations was hybridized to an Agilent Whole Human Genome Oligo Microarrays (8x60K v2, Design ID 039494)
2014-11-25 | E-GEOD-63197 | biostudies-arrayexpress
Project description:Memory B cells play a fundamental role in host defences against viruses. This dataset aimed at understanding their maturation and stability in the context of SARS-CoV-2 infection and consist of a longitudinal single-cell and repertoire profiling of the B cell response up to six months in four severe COVID-19 patients. All four patients were recruited at Henri Mondor University Hospital (AP-HP, Paris France), between March and May 2020, and required oxygen as treatment. Clinical and biological characteristics of these patients are summarized in the Patient_information.csv file. Peripheral (CD3-CD14-CD15-CD56-CD19+IgD-) B cells were FACS-sorted (MA900, Sony) in PBS/0.08% FCS from 4 patients (S-CoV) at baseline (M0) and 6 months (M6). 5x104 to 10x105 cells were obtained for each subset and 20000 were loaded in the 10x Chromium Controller to generate single-cell gel-beads in emulsion. The scRNA-seq libraries were generated using the Chromium Next GEM Single Cell V(D)J Reagent Kit v.1.1 with Feature Barcoding (10x Genomics) according to the manufacturer’s protocol. PBMCs were initially isolated from venous blood samples via standard density gradient centrifugation and used after cryopreservation at -150°C. Cells were thawed using RPMI-1640 (Gibco) 10% FBS, washed twice and incubated with 10µg of the SARS-CoV-2 his tagged spike protein in 100µL of PBS (Gibco) 2% FBS during 20 minutes on ice. Cells were washed and resuspended in the same conditions, then fluorochrome-conjugated antibody cocktail including the 2 anti-His was added at pre-titrated concentrations for 20 min at 4°C and viable cells were identified using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) incubated with conjugated antibodies used for cell sorting (CD3/CD14/CD15/CD56/CD19/IgD) as well as a panel of barcoded TotalSeqC® and homemade anti-His antibodies (see feature_reference.csv.gz files). Three distinct sorts were performed for each donor: two at the M0 time-point (M0_Sort1 and M0_Sort2) and one at the M6 time-point (M6_Sort1). His-tagged-Spike + barcoded-anti-his staining was only included in the last two sorts (M0_Sort2 and M6_Sort1). For these two sorts it should additionally be noted that 5’-transcriptomic and ADT libraries were sequenced in two separate runs (Run1 and Run2) to achieve sufficient sequencing depth for both libraries. Both runs were pooled at the Cell Ranger analysis step.
2021-01-31 | E-MTAB-9995 | biostudies-arrayexpress
Project description:Memory B cells play a fundamental role in host defenses against viruses. This dataset aimed at understanding the recruitment and remodeling of the memory B cell repertoire in the context of BA.1-breakthrough infection in BNT162b2 mRNA vaccinated individuals. All four donors enrolled in the study had received a booster (3rd dose) of BNT162b2 mRNA vaccine and had no history of prior SARS-CoV-2 infection. All four experienced a documented breakthrough infection between 12/24/2021 and 01/30/2022 when BA.1 was responsible for > 85% of SARS-CoV-2 infections in France. All donors were sampled at Henri Mondor University Hospital (AP-HP, Paris France), and samples used for scRNA-seq were collected shortly after breakthrough infection (PO_M0 samples; between day 7 and day 18) and 5 to 6 months after infection (PO_M6 samples; between day 152 and day 173). Clinical and biological characteristics of these patients are summarized in the Patient_information.csv file. For each sample, an initial pool of 50.000 total peripheral CD3-CD14-CD56- CD19+ IgD- cells was always sorted and afterward, to enrich for cells of interest, only CD19+CD38low antibody secreting cells (ASCs), CD19hi IgD+ cells and SARS-CoV-2 Spike/RBD PE/tetramer positive B cells were sorted, leading to approximately 55000-60000 total sorted cells per sample. Sorted cells were then counted and up to 20 000 cells were loaded in the 10x Chromium Controller to generate single-cell gel-beads in emulsion. The scRNA-seq libraries for gene expression (mRNA), ADT and VDJ BCR libraries were generated using Chromium Next GEM Single Cell V(D)J Reagent Kit v.1.1 with Feature Barcoding (10x Genomics) according to the manufacturer’s protocol. PBMCs were initially isolated from venous blood samples via standard density gradient centrifugation and used after cryopreservation at -150°C. PBMCs were thawed using RPMI-1640 (Gibco) 10% FBS, washed twice and approximately 15x106 cells were then resuspended in 100µL PBS 2%FBS and incubated for 40 minutes at 4°C with a decoy tetramer (biotinylated Bovine Serum albumin coupled with BV785 streptavidin) and Hu-1 Spike, BA.1 Spike, Hu-1 RBD and BA.1 RBD tetramers (constructed using PE-labelled TotalSeqC® streptavidin with different barcodes for each individual antigens (see feature_reference.csv.gz files).). Cells were washed, resuspended in 100µL PBS 2%FBS and stained with a cocktail of fluorochrome conjugated (CD3, CD14 both APC-H7 at 1:100 each; CD15 and CD56 BV785 at 1:100 each, CD19 PECF594 at 1:100, IgD FITC at 1:100, CD38 PercP-Cy5.5 at 1:100) and TotalSeqC® (CD38, CD27, CD71, CD21, CD11c, CD39, FCRL5, CD95 all at 1:40 (all obtained from BioLegend)) antibodies for 40 minutes on ice. Viable cells were identified using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific, 1:200) incubated with conjugated antibodies. Two distinct sorts were performed for each donor: one at the early time-point (PO_M0) and one at the 6 months’ time-point (PO_M6).
2023-08-15 | E-MTAB-12651 | biostudies-arrayexpress
Project description:B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a highly variable clinical course that reflects its heterogeneous genomic pattern. To better define molecular subtypes of the disease, we performed SNP and gene expression profiling microarray analyses in a panel of early stage (Binet A) patients. A clustering analysis of genomic profiles identified four significant groups mainly driven by del(13)(q14) and trisomy 12. Notably, patients with del(13)(q14) were grouped in two separate clusters based on the presence of a biallelic loss and the extension of the deletion. The shorter monoallelic deleted 13q14 region was found to be 635 kb in length, not encompassing the mir-15a/16-1 locus. Interestingly, the mir-15a and mir-16 expression was found to be significantly down-regulated only in patients with biallelic loss. Furthermore, a multiclass supervised analysis identified a different transcriptional signatures in the two genomic subgroups with del(13)(q14). Finally, an integrative approach identified 93 transcripts, mainly mapped to chromosome 12 and 13q12-q14.3, whose expression was significantly correlated with the DNA copy number. Overall, our data further support the notion that transcription deregulation in B-CLL could be mostly due to a gene dosage effect and underscore the presence of two distinct molecular types of 13q14 deleted patients with potential clinical relevance. Transcriptional analysis of 60 B-CLL patients, and genotyping analysis of 100 B-CLL patients, in early stage disease (Binet A). This series of microarray experiments contains the gene expression profiles of purified B-cell chronic lymphocytic leukemia (B-CLL) cells obtained from 60 newly diagnosed patients (Binet stage A). Peripheral blood mononuclear cells from B-CLL patients were isolated by Ficoll-Hypaque density-gradient centrifugation and the proportion of CD5/CD19/CD23 triple positive B cells in the suspension was determined by direct immunofluorescence performed using a FACS-sort flow cytometer with antibodies to: CD19 FITC/PE, CD23 PE and CD5 Cy-Chrome. If B-CLL cells were less than 90%, T cells, NK cells and monocytes were removed by negative selection using CD3, CD56, CD16, and CD14 monoclonal antibody treatment followed by magnetic beads. 3 micrograms of total RNA was processed and, in accordance with the manufacturer's protocols, 15 micrograms of fragmented biotin-labelled cRNA were hybridized on GeneChip Human Genome U133A Arrays (Affymetrix Inc.). The arrays were scanned using the GeneChip Scanner 3000 7G. The images were acquired using Affymetrix GeneChip Operating Software (GCOS version 1.4) and the probe level data converted to expression values using the Bioconductor function for the Robust Multi-Array average (RMA) procedure (Irizarry et al, 2003), in which perfect match intensities are background adjusted and quantile-quantile normalised. This series of microarray experiments contains the genome-wide profiles of purified B-cell chronic lymphocytic leukemia (B-CLL) cells obtained from 100 newly diagnosed patients (Binet stage A). Peripheral blood mononuclear cells from B-CLL patients were isolated by Ficoll-Hypaque density-gradient centrifugation and the proportion of CD5/CD19/CD23 triple-positive B cells in the suspension was determined by direct immunofluorescence performed using a FACS-sort flow cytometer with antibodies to: CD19 FITC/PE, CD23 PE and CD5 Cy-Chrome. If B-CLL cells were less than 90%, T cells, NK cells and monocytes were removed by negative selection using CD3, CD56, CD16, and CD14 monoclonal antibody treatment followed by magnetic beads. 250 nanograms of genomic DNA was processed and, in accordance with the manufacturer's protocols, 90 micrograms of fragmented biotin-labeled DNA were hybridized on GeneChip Human Mapping 250K NspI Arrays (Affymetrix Inc.). The arrays were scanned using the GeneChip Scanner 3000 7G. The images were acquired using Affymetrix GeneChip Operating Software (GCOS version 1.4). The raw data for individual SNPs were extracted from CEL files and converted into signal intensities using GTYPE 4.1 and Affymetrix Copy Number Analysis Tool (CNAT 4.0.1) softwares using the Hidden Markov Model algorithm with a genomic smoothing window set to 0. After the pre-processing, piecewise constant estimates of the underlying local DNA copy number (CN) variation was calculated using the DNAcopy Bioconductor package, which looks for optimal breakpoints using circular binary segmentation (CBS).
2010-09-01 | E-GEOD-16746 | biostudies-arrayexpress
Project description:Distinct genetic abnormalities such as TP53 deletion at 17p13.1, have been identified as having an adverse prognostic relevance in B-cell chronic lymphocytic leukemia (B-CLL). Conventional cytogenetic studies have shown that TP53 deletion in B-CLL is associated predominantly with 17p loss resulting from complex chromosomal rearrangements. We performed genome-wide DNA (SNPs arrays), fluorescence in situ hybridization (FISH) and gene expression profiling (GEP) analyses to investigate the significance of 17p loss in a panel of 71 genetically well-characterized B-CLLs in Binet stage A, 18 of which carried a TP53 monoallelic deletion. Combined SNP arrays and FISH approaches showed 17p loss in all of the TP53-deleted cases, with breakpoints scattered along the 17p11.2 region. Mutations in exons 5 to 9 of TP53 were found in 9/12 deleted samples. GEP of 60 B-CLLs, including 7 patients with 17p loss, identified 40 differentially expressed genes in 17p- versus 17p normal samples, 35 of which were down-regulated in 17p- tumors. The majority (30/35) of these transcripts, including putative tumor suppressor genes, mapped to 17p. Overall, these data indicate that, beside TP53 deletion, the concomitant loss of 17p arm may contribute to the strong negative prognostic impact known to be associated with this lesion in B-CLL. Keywords: transcriptional analysis of B-CLL with 17p loss patients This series of microarray experiments contains the gene expression profiles of purified B-cell chronic lymphocytic leukemia (B-CLL) cells obtained from 60 newly diagnosed patients (Binet stage A). Peripheral blood mononuclear cells from B-CLL patients were isolated by Ficoll-Hypaque density-gradient centrifugation and the proportion of CD5/CD19/CD23 triple positive B cells in the suspension was determined by direct immunofluorescence performed using a FACS-sort flow cytometer with antibodies to: CD19 FITC/PE, CD23 PE and CD5 Cy-Chrome. If B-CLL cells were less than 90%, T cells, NK cells and monocytes were removed by negative selection using CD3, CD56, CD16, and CD14 monoclonal antibody treatment followed by magnetic beads. 3 micrograms of total RNA was processed and, in accordance with the manufacturer's protocols, 15 micrograms of fragmented biotin-labelled cRNA were hybridized on GeneChip Human Genome U133A Arrays (Affymetrix Inc.). The arrays were scanned using the GeneChip Scanner 3000 7G. The images were acquired using Affymetrix GeneChip® Operating Software (GCOS version 1.4) and the probe level data converted to expression values using the Bioconductor function for the Robust Multi-Array average (RMA) procedure (Irizarry et al, 2003), in which perfect match intensities are background adjusted and quantile-quantile normalised.
2008-11-11 | E-GEOD-9992 | biostudies-arrayexpress
Project description:Distinct genetic abnormalities such as TP53 deletion at 17p13.1, have been identified as having an adverse prognostic relevance in B-cell chronic lymphocytic leukemia (B-CLL). Conventional cytogenetic studies have shown that TP53 deletion in B-CLL is associated predominantly with 17p loss resulting from complex chromosomal rearrangements. We performed genome-wide DNA (SNPs arrays), fluorescence in situ hybridization (FISH) and gene expression profiling (GEP) analyses to investigate the significance of 17p loss in a panel of 71 genetically well-characterized B-CLLs in Binet stage A, 18 of which carried a TP53 monoallelic deletion. Combined SNP arrays and FISH approaches showed 17p loss in all of the TP53-deleted cases, with breakpoints scattered along the 17p11.2 region. Mutations in exons 5 to 9 of TP53 were found in 9/12 deleted samples. GEP of 60 B-CLLs, including 7 patients with 17p loss, identified 40 differentially expressed genes in 17p- versus 17p normal samples, 35 of which were down-regulated in 17p- tumors. The majority (30/35) of these transcripts, including putative tumor suppressor genes, mapped to 17p. Overall, these data indicate that, beside TP53 deletion, the concomitant loss of 17p arm may contribute to the strong negative prognostic impact known to be associated with this lesion in B-CLL. Keywords: genomic analysis of B-CLL with 17p loss patients This series of microarray experiments contains the genome-wide profiles of purified B-cell chronic lymphocytic leukemia (B-CLL) cells obtained from 12 newly diagnosed patients (Binet stage A). Peripheral blood mononuclear cells from B-CLL patients were isolated by Ficoll-Hypaque density-gradient centrifugation and the proportion of CD5/CD19/CD23 triple positive B cells in the suspension was determined by direct immunofluorescence performed using a FACS-sort flow cytometer with antibodies to: CD19 FITC/PE, CD23 PE and CD5 Cy-Chrome. If B-CLL cells were less than 90%, T cells, NK cells and monocytes were removed by negative selection using CD3, CD56, CD16, and CD14 monoclonal antibody treatment followed by magnetic beads. 250 nanograms of genomic DNA was processed and, in accordance with the manufacturer's protocols, 40 micrograms of fragmented biotin-labelled DNA were hybridized on GeneChip Human Mapping 50K XbaI Arrays (Affymetrix Inc.). The arrays were scanned using the GeneChip Scanner 3000 7G. The images were acquired using Affymetrix GeneChip® Operating Software (GCOS version 1.4). Copy number values for individual SNPs were extracted and converted from CEL files into signal intensities using GTYPE 4.1 and Affymetrix Copy Number Analysis Tool (CNAT 4.0.1) softwares. Genomic Smoothing analysis was performed by using the smoothing window of 1 Mb, and inferred copy number states were derived from a Hidden Markov Model (HMM) based algorithm implemented in CNAT 4.0.1.
2008-11-11 | E-GEOD-11036 | biostudies-arrayexpress