Project description:Advances in Next Generation Sequencing (NGS) have made available a wealth of information that had previously been inaccessible to researchers and clinicians. NGS has been applied to understand genomic, transcriptomic, and epigenomic changes and gained traction as a significant tool capable of accelerating diagnosis, prognosis, and biomarker discovery. However, these NGS assays have yet to be practical methods for patient stratification or diagnosis because of the gap between the tiny quantities of biomaterials provided by a clinical sample and the large DNA input required by most of these assays. Current library preparation methodologies typically require large input amounts of DNA and a long and complicated manual process. Here we present a microfluidic reactor system for NGS library preparation, capable of reducing the number of pipetting steps significantly, automating much of the process, while supporting extremely low DNA input requirement (10 pg per library). This largely automated technology will allow for low-input preparations of 8 libraries simultaneously while reducing batch to batch variation and operator hands-on time.
Project description:We developed MHC1-TIP: an optimized mild acid elution-based immunopeptidomics workflow that leverages data independent acquisition and the Astral mass analyzer to enable scalable, single-tube and cost-effective MHC-I ligandome profiling. This dataset includes immunopeptidomics and proteomics data from the A375 melanoma cell line, colorectal cancer patient-derived organoids, and renal cell carcinoma tumour fragments. We show the effects of TGF-beta treatment on the proteome and immunopeptidome of A375 cells and interferon-gamma treatment on patient-derived organoids. We also explore immunopeptidome and proteome heterogeneity in patient-derived tumour fragments. MB_30: A375 immunopeptidomes in DDA (wildtype and B2M knockout); MB_38: Proteomes with and without mild acid treatment with A375 cells; MB_39: Proteomes and immunopeptidomes from TGFb treated A375 cells; MB_41: MHC1-TIP and MHC-I immunoprecipitation data from different A375 cell input numbers; MB_43: immunopeptidomes of A375 cells treated with different doses of interferon-gamma; MB_45: immunopeptidomes of A375 cells with MHC1-TIP and immunoprecipitation after mild acid elution to identify intracellular peptides; MB_46: Proteomics and immunopeptidomics data from 15 ex-vivo cultured patient-derived tumour fragments; MB_49: Proteomics and immunopeptidomics data from 3 patient-derived organoid lines.
Project description:we describe a streamlined RNAseq protocol (EASY RNAseq) for sensitive transcriptome assessment starting from low amount of input materials. EASY RNAseq is technically robust enough for sequencing small pools of homogenous and heterogeneous cells, recovering higher numbers of genes and with a more even expression distribution pattern than other commonly used methods.