Project description:Accurate Protein Characterization and Quantification of Xenograft Tumors Requires Separating Human and Mouse Cells prior to Proteomics Profiling
Project description:We developed a scalable assay permitting the simultaneous quantification of hundreds of proteins and the full transcriptome in thousands of individual cells from samples where cell number is limiting. The RNA Expression and Protein Sequencing assay (REAP-seq) uses DNA-labeled antibodies to permit DNA sequencing of both mRNA and antibodies in a single workflow using droplet microfluidics. We describe the development and validation of REAP-seq in human PBMCs, and use the assay to assess the costimulatory effects of an anti-CD27 agonist antibody in naïve CD8+ lymphocytes. Protein quantification using REAP-seq was more sensitive than parallel mRNA measurements and differentiated cell states with fewer analytes. Unbiased profiling of single cells without prior selection enabled identification and characterization of an unexpected cell type that would have been missed with population-averaged profiling methodologies.
Project description:Implantation is a milestone event during mammalian embryogenesis. Due to the extreme difficulty of obtaining in vivo human early post-implantation embryos, the gene regulatory network and epigenetics controlling human embryo implantation remains elusive. Here, combining an in vitro culture system for human post-implantation development and single-cell omics sequencing technologies, over 10,000 single cells at five representative stages of pre/post-implantation development were systematically analyzed. Unsupervised dimensionality reduction and clustering algorithm of the transcriptome data show stepwise implantation routes for the epiblast, primitive endoderm, and trophectoderm lineages, suggesting preparation for the establishment of a mother-to-offspring connection after implantation. Female embryos showed asynchronous progress of dosage loss of X chromosomes during implantation. Furthermore, using the single cell trio-Seq (scTrio-Seq) strategy, re-methylation of the genomes of all the three lineages was unambiguously revealed. Surprisingly, the genome re-methylation of PE lineage were much slower than both EPI and TE lineages during the implantation process, indicating distinct methylome features between EPI and PE although both of which were derived from ICM. Collectively, our work paves the way for understanding the complex molecular mechanisms that regulate human embryo implantation, informing new insights and future efforts in early embryonic development and reproductive medicine.
Project description:Evaluation of iTRAQ and SWATH-MS for the quantification of proteins associated with insulin resistance in human duodenal biopsy samples
Project description:Members of the miR-371 cluster are of the earliest de novo synthesized by embryos and have important actions in mammalian reproduction. MiR-371a is secreted by embryos at the peri-implantation period and its release has been previously associated with poor embryo quality and lower implantation rates in women undergoing IVF. Here we investigated the potential biological role of miR-371a release in regards to embryonic implantation, and specifically the molecular effects that this miRNA exerts on the cells of the endometrial lining. Gene expression changes in endometrial cells upon miR-371a addition were therefore identified using large-scale genomic plattform that covers the whole human genome (Affymetrix).
