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SRP190212
Homo sapiens
14 Downloadable Samples
Illumina HiSeq 2500
Description
Difference in RNA content of different cell types introduces bias to gene expression deconvolution methods. If ERCC spike-ins are introduced into samples, predicted proportions of deconvolution methods can be corrected Overall design: Two cell types of distinctly different sizes and RNA per cell content: HEK cells and Jurkat cells were mixed in different proportions ensuring that each mixture contained total of one million cells. We sequenced RNA of the samples (including ERCC spike-in controls to 382 be able to control for the absolute RNA-concentration).
Publication Title
Complete deconvolution of cellular mixtures based on linearity of transcriptional signatures.
Sample Metadata Fields
Cell line, Subject
View Samples- Mus musculus
- 57 Downloadable Samples
Affymetrix Mouse Gene 1.0 ST Array (mogene10st)
Description
Type 1 diabetes (T1D) is an autoimmune disease triggered by T cell reactivity to protein antigens produced by the -cells. Here we present a chronological compendium of transcriptional profiles from islets of Langerhans isolated from non-obese diabetic (NOD) mice ranging from 2 wks up to diabetes and compared to controls. Parallel analysis was made of cellular components of the islets. Myeloid cells populated the islets early during development in all mouse strains. This was followed by a type I interferon signature detectable at 4-6 wks of age only in diabetes susceptible mice. Concurrently, CD4 T cells were found within islets, many in contact with intra-islet antigen presenting cells. Early cellular signs of islet reactivity were detected by six wks. By 8 wks, NOD islets contained all major leukocytes populations and an inflammatory gene signature. This work establishes the natural transcriptional signature of T1D and provides a resource for future research.
Publication Title
Defining the transcriptional and cellular landscape of type 1 diabetes in the NOD mouse.
Sample Metadata Fields
Specimen part
View Samples- Mus musculus
- 33 Downloadable Samples
- Illumina HiSeq 2500
Description
We examined the transcriptional profiles of macrophages that reside in the islets of Langerhans of NOD, NOD.Rag1-/-, and B6.g7 mice at three weeks of age. Islet macrophages expressed an activation signature with high expression of Tnf, Il1b, and MHC-II both at the transcript and protein levels. These features are common with barrier macrophages of the lung and gastrointestinal tract. Moreover, injection of lipopolysaccharide induced a rapid inflammatory gene expression, indicating that blood stimulants are accessible to the macrophages and that these macrophages can sense them. In NOD mice, the autoimmune process imparted an increased inflammatory signature, including elevated expression of chemokines, chemokine receptors, and an oxidative response. The elevated inflammatory signature indicates that the autoimmune program was active at the time of weaning. Thus, the macrophages of the islets of Langerhans are poised to mount an immune response even at steady state, while the presence of the adaptive immune system elevates their activation state. Overall design: We examined the transcriptional profiles of macrophages that reside in the islets of Langerhans of NOD, NOD.Rag1-/-, and B6.g7 mice at three weeks of age. Lung macrophages and pancreatic LN dendritic cells of NOD mice were also examined.
Publication Title
The islet-resident macrophage is in an inflammatory state and senses microbial products in blood.
Sample Metadata Fields
Age, Specimen part, Cell line, Subject
View Samples- Mus musculus
- 2 Downloadable Samples
- Illumina HiSeq 2000
Description
A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments at the immunoglobulin heavy chain (Igh) loci. The C57BL/6 murine Igh locus has over 100 functional VH gene segments that can recombine to a rearranged DJH. While the non-random usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question we conducted deep sequencing of 5’-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. ChIP-seq results for several histone modifications and RNA polymerase II binding, RNA-seq for sense and antisense non-coding germline transcripts, and proximity to CTCF and Rad21 sites were compared to the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and non-coding RNA in general, but having a CTCF site near their RSS is critical, suggesting that position near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have high levels of histone marks and non-coding RNA, which may compensate for their poorer RSS and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for of each the four domains that comprise this locus. Overall design: RNA was extracted from C57BL/6 RAG-/- pro-B cells using Trizol® (Life Technologies Corp., Carlsbad CA) and genomic DNA was eliminated using the genomic DNA wipeout buffer in the QuantiTect Reverse transcription kit (QIAGEN). A final purification of the RNA was performed with the RNeasy kit from QIAGEN. For each sample, 100 ng of total RNA was used to make RNASeq libraries using the NuGEN Encore Complete DR kits following manufacturer''s recommended protocols. Sequencing libraries were gel purified to ensure insert sizes were larger than 100 bp in length and sequenced on an Ilumina HiSeq2000 for 100 bases plus 7 bases for indexing.
Publication Title
Deep sequencing of the murine IgH repertoire reveals complex regulation of nonrandom V gene rearrangement frequencies.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Mus musculus
- 17 Downloadable Samples
- Affymetrix Mouse Gene 1.0 ST Array (mogene10st)
Description
These studies utilized two TCR transgenic mouse lines, LLO118 and LLO56, that our laboratory has developed and characterized, which recognize the same Listeria monocytogenes LLO/IO-Ab epitope with equal affinities. When 104 naive CD4+ LLO T cells are transferred into a B6 mouse and one day later infected with wild type Listeria monocytogenes, the LLO118 T cells have a more robust primary expansion than LLO56. In contrast, after a secondary challenge, LLO56 T cells have a much greater expansion than LLO118 T cells. One striking phenotypic difference between the LLO118 and LLO56 T cells lies in their CD5 expression. CD5 expression has been shown to correlate directly with TCR affinity for self-pMHC and tonic signaling. LLO56 cells have a higher basal phosphorylation of the TCR chain, and they have significantly increased expression of Nur77 mRNA. These transcriptional profiling experiments examined if there were transcriptional differences between LLO118 and LLO56 T cells, either naive or after D7 of infection, that would account for their disparate in vivo behaviors.
Publication Title
Tonic TCR Signaling Inversely Regulates the Basal Metabolism of CD4<sup>+</sup> T Cells.
Sample Metadata Fields
Specimen part
View Samples- Mus musculus
- 6 Downloadable Samples
- Illumina HiSeq 2500
Description
mRNA expression in Eµ-Myc lymphoma cells expressing or lacking miR-17~92 Overall design: Eµ-Myc B-cell lymphomas harboring conditional alleles of miR-17~92 were cultured with or without 4-OHT to generate isogenic tumour cells with homozygous deletion of miR-17~92. Wild type (fl) and miR-17~92-deleted (del) Eµ-Myc cells were cultured for 48 hours under regular growth conditions, and RNA isolated for sequencing analysis.
Publication Title
The miR-17∼92 microRNA Cluster Is a Global Regulator of Tumor Metabolism.
Sample Metadata Fields
Specimen part, Subject
View Samples- Mus musculus
- 18 Downloadable Samples
- Illumina HiSeq 2500
Description
Immune-Responsive Gene 1 (Irg1) is a mitochondrial enzyme that produces itaconate under inflammatory conditions principally in cells of myeloid lineage. Cell culture studies suggest that itaconate regulates inflammation through inhibitory effects on cytokine and reactive oxygen species production. To evaluate the functions of Irg1 in vivo, we challenged wild-type (WT) and Irg1 KO mice with Mycobacterium tuberculosis (Mtb) and monitored disease progression. Irg1 KO but not WT mice succumbed rapidly to Mtb, and mortality was associated with increased infection, inflammation, and pathology. Infection of LysM-Cre Irg1 flox, MPR8-Cre Irg1 flox, and CD11c-Cre Irg1 flox conditional knockout mice along with neutrophil depletion experiments revealed a role for Irg1 in alveolar macrophages and LysM+ myeloid cells in preventing neutrophil-mediated immunopathology and disease. RNA-seq analyses suggest that Irg1 and its production of itaconate temper Mtb-induced inflammatory responses in myeloid cells at the transcriptional level. Thus, Irg1 modulates inflammation to curtail Mtb-induced lung disease. Overall design: Neutrophils were purified from bone marrow of naïve mice by negative selection using magnetic-activated cell sorting beads (Miltenyi). Neutrophil purity (>95%) was assessed by flow cytometry as the percentage of Ly6G+ CD11b+ cells. Neutrophils were cultured in RPMI-1640 supplemented with 1% non-essential amino acids at 37°C, 5% CO2. GFP-Mtb was grown to mid-log phase, washed with PBS, sonicated to disperse clumps, and resuspended in neutrophil culture media. GFP-Mtb then was opsonized prior to infection by mixing with an equal volume of normal mouse sera (Sigma) and incubation at room temperature for 30 min. Neutrophils were mock-infected or infected with opsonized GFP-Mtb at MOI 1 and incubated at 37°C, 5% CO2.
Publication Title
<i>Irg1</i> expression in myeloid cells prevents immunopathology during <i>M. tuberculosis</i> infection.
Sample Metadata Fields
Specimen part, Cell line, Subject, Time
View Samples- Mus musculus
- 15 Downloadable Samples
- Illumina HiSeq 2500
Description
Immune-Responsive Gene 1 (Irg1) is a mitochondrial enzyme that produces itaconate under inflammatory conditions principally in cells of myeloid lineage. Cell culture studies suggest that itaconate regulates inflammation through inhibitory effects on cytokine and reactive oxygen species production. To evaluate the functions of Irg1 in vivo, we challenged wild-type (WT) and Irg1-/- mice with Mycobacterium tuberculosis (Mtb) and monitored disease progression. Irg1-/- but not WT mice succumbed rapidly to Mtb, and mortality was associated with increased infection, inflammation, and pathology. Infection of LysM-Cre Irg1fl/fl, MPR8-Cre Irg1fl/fl, and CD11c-Cre Irg1fl/fl conditional knockout mice along with neutrophil depletion experiments revealed a role for Irg1 in alveolar macrophages and LysM+ myeloid cells in preventing neutrophil-mediated immunopathology and disease. RNA-seq analyses suggest that Irg1 and its production of itaconate temper Mtb-induced inflammatory responses in myeloid cells at the transcriptional level. Thus, Irg1 modulates inflammation to curtail Mtb-induced lung disease. Overall design: Macrophages were obtained by culturing bone marrow cells in RPMI-1640 (Invitrogen) supplemented with 10% heat inactivated fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids, 100 U penicillin per mL, 100 µg streptomycin per mL, and 22 ng M-CSF (Peprotech) per ml for 6 days at 37°C, 5% CO2. Fresh media was added on day 3 of culture. After 6 days of culture, non-adherent cells were discarded. Adherent macrophages were switched into antibiotic-free media and seeded at 105 cells per well and 9 x 105 cells per well in tissue culture-treated 96 and 6 well plates respectively. In some cases, macrophages were treated with 0.25 mM itaconic acid (Sigma) for 12 h prior to inoculation with Mtb. Mtb was grown to mid-log phase, washed with PBS, sonicated to disperse clumps, and resuspended in antibiotic-free macrophage culture media. Macrophage cultures were inoculated by adding Mtb-containing media at a multiplicity of infection (MOI) of 1 and centrifuging for 10 min at 200 x g. Cells were washed twice with PBS to remove unbound Mtb, fresh culture media was added, and cells were incubated at 37°C, 5% CO2. In some cases culture media was supplemented with 0.25 mM itaconic acid.
Publication Title
<i>Irg1</i> expression in myeloid cells prevents immunopathology during <i>M. tuberculosis</i> infection.
Sample Metadata Fields
Specimen part, Treatment, Subject
View Samples- Mus musculus
- 11 Downloadable Samples
- Illumina HiSeq 2500
Description
Immune-Responsive Gene 1 (Irg1) is a mitochondrial enzyme that produces itaconate under inflammatory conditions principally in cells of myeloid lineage. Cell culture studies suggest that itaconate regulates inflammation through inhibitory effects on cytokine and reactive oxygen species production. To evaluate the functions of Irg1 in vivo, we challenged wild-type (WT) and Irg1 KO mice with Mycobacterium tuberculosis (Mtb) and monitored disease progression. Irg1 KO but not WT mice succumbed rapidly to Mtb, and mortality was associated with increased infection, inflammation, and pathology. Infection of LysM-Cre Irg1 flox, MPR8-Cre Irg1 flox, and CD11c-Cre Irg1 flox conditional knockout mice along with neutrophil depletion experiments revealed a role for Irg1 in alveolar macrophages and LysM+ myeloid cells in preventing neutrophil-mediated immunopathology and disease. RNA-seq analyses suggest that Irg1 and its production of itaconate temper Mtb-induced inflammatory responses in myeloid cells at the transcriptional level. Thus, Irg1 modulates inflammation to curtail Mtb-induced lung disease. Overall design: C57BL/6N (WT) mice were purchased from Charles River. B6.SJL (CD45.1) mice were obtained from Jackson Laboratories. Irg1-/- mice (embryonic stem cells obtained from KOMP (C57BL/6N background), MGI: 103206) were generated at Washington University. Adult mice (6-13 weeks of age) of both sexes were used, and sex was randomized between experiments. Neutrophils were purified by magnetic-activated cell sorting from the bone marrow of naïve mice (negative selection) or the lungs of Mtb-infected mice at 16 dpi (selection for Ly6G+ cells) (Miltenyi).
Publication Title
<i>Irg1</i> expression in myeloid cells prevents immunopathology during <i>M. tuberculosis</i> infection.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Mus musculus
- 4 Downloadable Samples
- Illumina HiSeq 2500
Description
Using complementary forms of high dimensional profiling we define differences in CD45+ cells from syngeneic mouse tumors that either grow progressively or eventually reject following immune checkpoint therapy (ICT). Unbiased assessment of gene expression of tumor infiltrating cells by single cell RNA sequencing (scRNAseq) and longitudinal assessment of cellular protein expression by mass cytometry (CyTOF) revealed significant remodeling of both the lymphoid and myeloid intratumoral compartments. Surprisingly, we observed multiple subpopulations of monocytes/macrophages distinguishable by the combinatorial presence or absence of CD206, CX3CR1, CD1d and iNOS, markers of different macrophage activation states that change over time during ICT in a manner partially dependent on IFN?. Both the CyTOF data and additional analysis of scRNAseq data support the hypothesis that macrophage polarization/activation results from effects on circulatory monocytes/early macrophages entering tumors rather than on pre-polarized mature intratumoral macrophages. Thus, ICT induces transcriptional and functional remodeling of both myeloid and lymphoid compartments. Overall design: Droplet-based 3' end massively parallel single-cell RNA sequencing was performed by encapsulating sorted live CD45+ tumor infiltrating cells into droplets and libraries were prepared using Chromium Single Cell 3' Reagent Kits v1 according to manufacturer's protocol (10x Genomics). The generated scRNAseq libraries were sequenced using an Illumina HiSeq2500.
Publication Title
High-Dimensional Analysis Delineates Myeloid and Lymphoid Compartment Remodeling during Successful Immune-Checkpoint Cancer Therapy.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples