Project description:Functional discovery of tumor-reactive T cell receptors by massively parallel library synthesis and screening: NKIRTIL063 titration screen
| PRJNA1068301 | ENA
Project description:Functional discovery of tumor-reactive T cell receptors by massively parallel library synthesis and screening: NKIRTIL063 neoantigen screen
| PRJNA1068303 | ENA
Project description:Functional discovery of tumor-reactive T cell receptors by massively parallel library synthesis and screening: validation in CD4 T cells and OVC190 TCR screen
Project description:Purpose: Determine the change in expression of genes in AR deficient CD8 T cells Methods: C57Bl/6J splenocytes were isolated, total CD8 T cells magnetically enriched and electroporated with non-targeting gRNA or AR-specific gRNAs. Cells were stimulated with aCD3/28 for three days before isolating RNA and library prep. Sequencing was performed by the Massively Parallel Sequencing Shared Resource (MPSSR) at OHSU. Results: AR regulated genes in mouse CD8 T cells.
Project description:The screening of a previously reported fluorescein labelled 10,000 member PNA encoded peptide library allowed information on the interaction between the peptide-ligands and the cell surface receptors to be extracted, identified new peptide ligands for cell surface receptors, and gave crucial information about consensus sequences. A novel indirect amplification of the PNA signal by amplification of the PNA-complementary DNA library was developed to screen PNA-encoded peptide library against D54, HEK293T, and HEK293T-CCR6 cells. This work generates a new approach to biological discovery and an expansion of modern microarray techniques. In addition, the microarray approach facilitates screening for differences in surface-receptor ligands and/or receptor expression between various cell types including diseased and normal cells.
Project description:Here we developed a massively parallel in-library ligation methodology to simultaneously perturb four pre-designed targets in CRISPR/Cas9 screening. Thousands of pairs of sequences precisely ligated with their counterparts in library, which enabled simultaneous expression of four gRNAs from each single vector. We demonstrated this novel method with 6,236 4-gene combinations targeting 1,599 immune response related genes, and generated a plasmid library with 1,400x coverage. The library performance was evaluated in a canonical T cell activation experiment, and combinations involved in TCR signaling pathway or TCR complex were successfully identified as positive regulators. Novel combination that is reflecting a potential pathway crosstalk was also verified. This new methodology expands the capacity of the perturbation in CRISPR screening and provided a powerful tool for researches in broad fields to study the combinatorial outcomes from coordinated gene behaviors.
Project description:Homo sapiens and Macaca fascicularis neural progenitor cell lines were transduced with a lentiviral MPRA (Massively Parallel Reporter Assay) library. MPRA barcode sequencing and RNA-seq was performed on the extracted RNA. RNA-seq data was used to confirm transcription factor expression.
Project description:In this manuscript we describe our work on the development of a label-free chemoproteomics screening platform for cysteine reactive covalent fragments on a 96 well plate format. Within this workflow, we profile cysteine reactive fragments by competition with the hyper-reactive iodoacetamide desthiobiotin (IA-DTB) in cell lysates and live cells. We employ label free quantification and data independent acquisition (DIA) on an Evosep One – Bruker timsTOF Pro. The platform was used to screen a library of 80 Cys-reactive covalent fragments in HEK293T and Jurkat cell lysate, identifying functionally relevant hit fragments for numerous cysteine sites and demonstrating the utility of the approach in expanding the ligandable proteome. We have also validated a number of selective interactions through dose response and further expanded our proteomics platform to perform hit expansion studies and in-cell validation.