Project description:Flow cytometry sorted B-cells reactive to ACE2 peptides isolated from peripheral blood of COVID-19 patients compared with non-reactive B-cells using pooled hashtag barcoding and 10x genomics 5'DGE kit and VDJ recombination of B-cells
Project description:We measured gene expression in single-cell RNA sequencing samples from patients with high-grade serous ovarian cancer (HGSOC), for a study on improving HGSOC subtype definition by taking into account varying cell type proportions within tumors.
Project description:Tumor cells were isolated from solid tumors or ascites from HGSOC patients and enriched for EpCAM expression by antibody magnetic beads (affinity)-based methods and large RNAs (> 200 nt) sequenced (50 bp paired ends) after rRNA depletion (RiboZero). Expression differences between patients with miliary and non-miliary peritoneal tumor spreading were used for a search for new targeted therapies and for biological annotion.
Project description:In order to investigate heterogeneity and dynamics of cancer-associated immune cells, we performed single-cell sequencing on peripheral and tumor-infiltrating immune cells in three renal clear cell carcinoma patients. We chose renal clear cell carcinoma tumors based on the responsive of these tumors to immune checkpoint blockade in the context of low mutational loads, which implies a strong influence from the tumor microenvironment. Using the 10x Genomic 5 expression platform a total of 25,672 immune cells were isolated and passed filtering for quality control, with 13,433 cells from peripheral blood and 12,239 tumor-infiltrating cells. In addition, we used the Chromium Single Cell V(D)J kit to enrich for T cell receptor sequences for nearly 10,000 T cells in with accompanying expression information, allowing for the investigation of clonality and transcriptional phenotypic diversity. We generated a comprehensive immune profile using single-cell RNA-seq data from 25,000 immune cells from three renal cell carcinoma along with matched peripheral blood. In addition, we performed VDJ sequencing on the isolated single T cells.
Project description:CD4 T cells are recruited to the FRT following Chlamydia intravaginal infection. We use single-cell RNA sequencing and VDJ profiling to compare CD4 T cells sorted from WT (B6) and Bhlhe40-/- mice at 14 days post Chlamydia muridarum intravaginal infection.
Project description:For high-throughput sequencing and quantification of immunoglobulin repertoires, most methodologies utilise RNA. However, output varies enormously between recombined genes due to different promoter strengths and differential activation of lymphocyte subsets, precluding quantitation of recombinants on a per cell basis. To date, DNA-based approaches have used V gene primer cocktails, with substantial inherent biases. Here we describe VDJ-seq, which accurately quantitates immunoglobulin diversity at the DNA level in an unbiased manner. This is accomplished with a single primer extension step using biotinylated J gene primers. By addition of unique molecular identifiers (UMI) before primer extension, we reliably remove duplicate sequences and correct for sequencing and PCR errors. Furthermore, VDJ-seq captures productive and non-productive VDJ and DJ recombination events on a per cell basis. Library preparation takes 3 days, with 2 days of sequencing, and 1 day of data processing and analysis.