Project description:Highly-multiplexed immunohistochemistry (mIHC) enables the staining and quantification of dozens of antigens in a tissue section with single-cell resolution. However, annotating cell populations that differ little in the profiled antigens or for which the antibody panel does not include specific markers is challenging. To overcome this obstacle, we have developed an approach for enriching mIHC images with single-cell RNA-seq data, building upon recent experimental procedures for augmenting single-cell transcriptomes with concurrent antigen measurements. Spatially-resolved Transcriptomics via Epitope Anchoring (STvEA) performs transcriptome-guided annotation of highly-multiplexed cytometry datasets. It increases the level of detail in histological analyses by enabling annotation of subtle cell populations, spatial patterns of transcription, and interactions between cell types. More generally, it enables the systematic annotation of cell populations in cytometry data. We demonstrate the utility of STvEA by uncovering the architecture of poorly characterized cell types in the murine spleen using published highly-multiplexed cytometry and mIHC data.
Project description:Bulked segregant analysis (BSA) is an efficient method to rapidly and efficiently map genes responsible for mutant phenotypes. This procedure, however, requires access to quantitative genetic markers that are polymorphic in the mapping population. We have developed a modification of BSA (BSR-Seq) that makes use of RNA-Seq reads to efficiently map genes even in populations for which no polymorphic markers have been previously identified. Because of the digital nature of next-generation sequencing (NGS) data, it is possible to conduct de novo SNP discovery and quantitatively genotype BSA samples using the same RNA-Seq data. In addition, analysis of the RNA-Seq data provides information on the effects of the mutant on global patterns of gene expression at no extra cost. In combination these results greatly simplify gene cloning experiments. To demonstrate the utility of this strategy BSR-Seq was used to clone the glossy3 (gl3) gene of maize. Mutants of the glossy loci exhibit altered accumulation of epicuticular waxes on juvenile leaves. We previously generated a large collection of glossy mutants using the Mu transposon system. By subjected a reference allele to BSR-Seq, we were able to map the gl3 locus to a ~2.3Mb interval that is consistent with the results of prior mapping experiments. The single gene located in the 2.3Mb mapping interval that contained a Mu insertion and whose expression was down-regulated in the mutant pool was subsequently demonstrated to be the gl3 gene via the analysis of multiple independently Mu transposon induced mutant alleles. The gl3 gene encodes a putative myb transcription factor, which directly or indirectly affects the expression of a number of genes involved in the biosynthesis of very-long-chain fatty acids.
Project description:We used scRNA-seq data generated using Smart-seq2 to reconstruct the native TCR from Ag-specific T cells and then to link these with the gene expression profile of individual cells. antigen specific CD8 T cells were obtained from a subject who had previously cleared hepatitis C virus (HCV) infection. A cell line was also generated from the same subject and included in the analysis with and without antigen stimulation.