Project description:SORT-seq is a plate-based method of single-cell RNA sequencing (Muraro et al. 2016) is a partially robotized version of the CEL-seq2 protocol (Hashimshony et al. 2016). Synovial membranes from Rheumatoid arthritis' patients yield a relatively robust number of DCs using scRNAseq, we sought to validate this initial finding at protein level. We employed a rigorous gating strategy to sort specific ST DC subsets, developed based on CITEseq analysis, which was then confirmed by index cell plate sort sequencing (SORT-seq).
Project description:Methods: Cells isolated from the whole brains of naive wild type, infected wild type, and infected CCR2-DTR mice were sort-purified on live CD45hi cells from five biological replicates and pooled. Sort-purified cells were then processed using the 10X Genomics Chromium Controller. Cell suspensions were loaded onto the Chromium Single Cell A Chip for cell lysis and barcoding. RNA from individual cells was reverse transcribed and sequencing libraries prepared using the Chromium Single Cell 3’ Library Kit v2 following the manufacturers protocol. Samples were sequenced using an Illumina NextSeq 550 with standard 10X Genomics Configuration (26 bp x 98 bp). After sequencing, raw bcl files were processed using the cellranger mkfastq command for sample demultiplexing and conversion to .fastq files, followed by cellranger count for cell barcode and UMI deconvolution as well as mapping to the respective reference genome. Processed digital gene expression matrices were imported into R studio for analysis using the Seurat package. Samples were aligned along common sources of variation and compared using canonical correlation analysis to identify unique clusters of cells within the samples. Marker genes for each sample and cluster were identified and used for generation of downstream plots within the Seurat package. All packages are maintained to be best in class and are regularly updated to their most recent release. Results: 10 distinct cell clusters were identified and we found that two populations were found at increased proportions post-infection but were substantially reduced in the monocyte-depleted animals (CCR2-DTR). We confirmed that these populations to be monocytes and monocyte-derived cells, and valicated their immunoregulatory molecules by RT-qPCR analysis.
Project description:We performed single-cell RNA sequencing (scRNA-seq) on infrarenal abdominal aortas from C57BL/6J mice after perivascular CaCl2 treatment. Infrarenal abdominal aortas were collected four days after AAA induction and processed for sequencing. These data provide high-resolution insight into the complexity and heterogeneity of mouse AAA.
Project description:We performed single-cell/nuclei RNA-sequencing (sc/snRNA-seq) of 22 treatment-naïve melanoma brain metastases (MBM; 5 samples using scRNA-seq and 17 snRNA-seq) from 21 patients and 10 treatment-naïve peripheral (extracranial) metastases (ECM; all snRNA-seq) from 10 patients. We performed matched spatial sequencing using SlideSeq2 (n=16) on 11 snRNA-seq samples.
Project description:Murine colorectal cancer cells were injected into the portal vein of NOD.Cg-PrkdcSCID Il2rgtm1Wjl/SzJ mice. Liver metastatic cells were harvested at specific time points for single-cell RNA-sequencing (SORT-seq).
Project description:Murine breast cancer organoids (MMTV-PyMT) were embeded in basement membrane extract (BME, invasion non-permissive) or collagen I (invasion-permissive). Organoids were harvested after 5days for single-cell RNA-sequencing (SORT-seq).