Project description:Gene expression and T cell receptor profiles from single cells from populations of T cells stimulated with islet autoantigenic peptides. CD4 T cells were stimulated with native (proinsulin, GAD fragments) or neo-antigen epitopes or flu vaccine (Pediacel). Activated cells (identified as CD19- CD3+ CD4+,CD45-RO+ CD95+, CD154+CD69+ CD27+) were FACS-sorted, barcodes for samples were introduced with cell hashing as follows: patient, 1 pool 1 (Hashtag 1); patient 1 pool 2 (Hashtag 2) patient 2 pool 1 (Hashtag 3); patient 2 pool 2 (Hashtag 4) and Pediacel (patient 1 and 2; Hashtag 5). Hashtag info in ADT files, TCR sequences provided in VDJ files.
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.
Project description:Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with currently no effective treatment. While half of MPNSTs arise sporadically, the others are due to the malignant progression of neurofibromas (NFs) in patients with neurofibromatosis type 1 (NF1). NFs are benign nerve sheath tumors due to bi-allelic loss of NF1, encoding a negative regulator of Ras pathway, in the Schwann cell (SC) lineage. We have conceived an Nf1-KO mouse model in which NFs spontaneously transform into MPNSTs To decipher the step-by-step evolution of the cellular composition and biological activities of tumor cells and their microenvironment during the transformation process, we performed single-cell transcriptomic profiling of pNFs, dyNFs and MPNSTs, all issued from Prss56Cre, Nf1fl/fl cohort, using 10x Chromium platform. We discovered that malignant transformation is initiated by a molecular transition of tumor SCs from glial to mesenchymal identity (GMT).
Project description:Differentiation of HES3 hESCs using BMP4, Activin A and CHIR99021. 5 timepoints of sample collection (day 3-12). Differentiations were started sequentially and collected and processed on the same day. Sample1 = day 3 + day3.75 (50:50 mix); Sample2 = day 4.75; Sample3 = day 5.75; Sample 4 = day 12.
Project description:Using COVID-19 as model, we set out to identify serological, cellular and transcriptomic imprints of pathological responses linked to autoreactive B cells at single-cell resolution
Project description:Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare disease caused by the expression of progerin, an aberrant protein produced by a de novo point mutation in the LMNA gene. HGPS patients show accelerated aging and die prematurely, mainly from atherosclerosis complications. Understanding vascular disease onset and progression in HGPS and uncovering new therapeutic targets critically depend on the identification of cell type-specific molecular and functional alterations in the highly heterogeneous cell subsets present in the arterial wall. We used single-cell RNA sequencing to characterize the cellular and molecular landscape of the aorta in progerin-expressing LmnaG609G/G609G mice and wild type controls. Subendothelial extracellular matrix (ECM) stiffness was analyzed in decellularized aortas by atomic force microscopy, and aortic blood flow in vivo was monitored by ultrasound assessment. For atherosclerosis studies, we used progeroid atheroprone Apoe–/– LmnaG609G/G609G mice.
Project description:The purpose of the experiment was to investigate the differentiation state gradient among antigen-experienced CD8+ T cells in healthy human blood. This was a sub-goal within a larger experiment, which aimed to investigate the clonal relationship between CD8+ T cells in blood and skin of matched donors. The blood T cells were sorted as CD8+ T cells (n=4) or CD3+ T cells (n=2), processed according to the 5´prime 10x genomic workflow, and sequenced at NGI Sweden.
Project description:Smooth muscle cells (SMCs) undergo phenotypic switching during atherosclerosis and give rise to a large fraction of plaque cells. To investigate the role of hypoxia-inducible factor 1α (HIF1α) signaling in SMC behaviour during atherogenesis, we used mice in which Hif1a is conditionally deleted in lineage-traced SMCs (Myh11-CreERT2; Rosa26-tdTomato; Hif1a-floxed). Cre recombination was induced with tamoxifen at 6 weeks of age. Atherosclerosis was induced by a single tail-vein injection of rAAV8-PCSK9 at 9 weeks of age followed by 21 weeks of high-fat diet feeding. Aortic arches were pooled by genotype from SMC-specific Hif1a knockout (Hif1aSMC-KO, n=8) and wild-type control (Hif1aWT, n=6) male mice, enzymatically dissociated, and viable cells (Draq5+/DAPI–) isolated by fluorescence-activated cell sorting. Single-cell transcriptomes were generated using the 10x Genomics Chromium Next GEM Single Cell 3′ v3.1 platform and sequenced on an Illumina HiSeq 4000. Reads were aligned to the mm10 reference transcriptome supplemented with the tdTomato transgene sequence using Cell Ranger. The dataset enables comparison of cell-type composition and of SMC-derived cell transcriptional states between genotypes, with a focus on hypoxia- and stress-response pathways underlying SMC phenotypic modulation. Only male mice were studied because the Myh11-CreERT2 transgene is Y-linked.
Project description:iPS cell derived neural stem cells and differentiated cells from one healthy individual and one indivudal carrying NRXN1-a biallelic deletion investigated with single cell RNA-sequencing. Celltype characterization and comparison revealed variations between stem cell identity and in cell differentiation outcome across the two individuals.
Project description:The aim of the experiment was to investigate the clonal relationship between CD8+ T cell subsets of donor matched blood with epidermal CD8+ T cells. Abdominal skin (n=8) was separated into dermis and epidermis, followed by epidermal skin and PBMC isolation. Single CD8+ T cells (n=6) and CD3+ T cells (n=2) were FACS sorted, processed with the 5’ 10x Genomics workflow and sequenced at NGI Sweden.