Project description:The development of human induced pluripotent stem cell (iPSC)–based regenerative therapies is challenged by the lack of specific cell markers to isolate differentiated cell types and improve differentiation protocols. This issue is particularly critical for notochordal-like cells and chondrocytes, which are crucial in treating back pain and osteoarthritis, respectively. Both cell types produce abundant proteoglycan aggrecan (ACAN), crucial for the extracellular matrix. We generated two human iPSC lines containing an ACAN-2A-mScarlet reporter. The reporter cell lines were validated using CRISPR-mediated transactivation and functionally validated during notochord and cartilage differentiation. The ability to isolate differentiated cell populations producing ACAN enables their enrichment even in the absence of specific cell markers and allows for comprehensive studies and protocol refinement. ACAN’s prevalence in various tissues (e.g., cardiac and cerebral) underscores the reporter’s versatility as a valuable tool for tracking matrix protein production in diverse cell types, benefiting developmental biology, matrix pathophysiology, and regenerative medicine.
Project description:These experiments use a barcoded pool of reporter transcripts, each of which encode the same mScarlet-PPIG_LCD fusion protein, but using different degrees of GA-multivalency via codon bias, and containing a different number of constitutive introns. In order to be able to perform experiments using this pool, it was necessary to perform long-read sequencing of the plasmid pool to relate the barcodes in the 3' ends of the reporter to their gene structure. Therefore, we performed long-read sequencing of the plasmid pool (both the original pool used for transfection and the ePB plasmid used for PiggyBac integration). Furthermore, to determine the splicing patterns of the reporter genes, we transfected the plasmid pool into HeLa cells for 16 hours, then performed targeted long-read sequencing of the reporter plasmids via RT-PCR. Note: the Nanopore adapter ligation strategy means that reads can come in either orientation. To determine the gene architectures and barcodes, we used fuzzy string matching. First we matched to various fixed sequences throughout the reporter transcripts to determine the orientation of the read and that the read spanned the full length of the transcript. Then we used the same string matching strategy to detect the presence of the different intronic or exonic sequences - the gene architecture. Then we extracted the associated unique plasmid barcode associated with that gene architecture. Example reporter sequences can be found here: https://benchling.com/faraway/f_/kXCfddtQ-public-reporter-plasmid-maps/ or alternatively, in Supplemental Table 2 of the bioRxiv submission here: https://www.biorxiv.org/content/10.1101/2023.08.21.554177v1.supplementary-material
Project description:To examine the protein spatial and temporal changes upon carfilzomib-mediated proteasome inhibition in cardiac cells, we produced human iPSC-derived cardiomyocytes using a standard small molecule based protocol. Cardiomyocyte identity was confirmed by morphology, observation of contraction, and the presence of GFP tagged MLC-2a in the reporter line. We then applied the SPLAT protocol to untreated and carfilzomib-exposed iPSC-cardiomyocytes.
Project description:To examine the protein spatial and temporal changes upon carfilzomib-mediated proteasome inhibition in cardiac cells, we produced human iPSC-derived cardiomyocytes using a standard small molecule based protocol. Cardiomyocyte identity was confirmed by morphology, observation of contraction, and the presence of GFP tagged MLC-2a in the reporter line. We then applied the SPLAT protocol to untreated and carfilzomib-exposed iPSC-cardiomyocytes.
Project description:a chromosome-level nuclear genome and organelle genomes of the alpine snow alga Chloromonas typhlos were sequenced and assembled by integrating short- and long-read sequencing and proteogenomic strategy
Project description:Objectives: To perform long-read transcriptome and proteome profiling of pathogen-stimulated peripheral blood mononuclear cells (PBMCs) from healthy donors. We aim to discover new transcripts and protein isoforms expressed during immune responses to diverse pathogens. Methods: PBMCs were exposed to four microbial stimuli for 24 hours: the TLR4 ligand lipopolysaccharide (LPS), the TLR3 ligand Poly(I:C), heat-inactivated Staphylococcus aureus, Candida albicans, and RPMI medium as negative controls. Long-read sequencing (PacBio) of one donor and secretome proteomics and short-read sequencing of five donors were performed. IsoQuant was used for transcriptome construction, Metamorpheus/FlashLFQ for proteome analysis, and Illumina short-read 3’-end mRNA sequencing for transcript quantification. Results: Long-read transcriptome profiling reveals the expression of novel sequences and isoform switching induced upon pathogen stimulation, including transcripts that are difficult to detect using traditional short-read sequencing. We observe widespread loss of intron retention as a common result of all pathogen stimulations. We highlight novel transcripts of NFKB1 and CASP1 that may indicate novel immunological mechanisms. In general, RNA expression differences did not result in differences in the amounts of secreted proteins. Interindividual differences in the proteome were larger than the differences between stimulated and unstimulated PBMCs. Clustering analysis of secreted proteins revealed a correlation between chemokine (receptor) expression on the RNA and protein levels in C. albicans- and Poly(I:C)-stimulated PBMCs. Conclusion: Isoform aware long-read sequencing of pathogen-stimulated immune cells highlights the potential of these methods to identify novel transcripts, revealing a more complex transcriptome landscape than previously appreciated.
Project description:To identify aberrant splicing isoforms and potential neoantigens, we performed full-length cDNA sequencing of lung adenocarcinoma cell lines using a long-read sequencer MinION. We constructed a comprehensive catalog of aberrant splicing isoforms and detected isoform-specific peptides using proteome analysis.
2020-12-09 | PXD019915 | JPOST Repository
Project description:iPSC Retinal Pigmented Epithelium short and long read sequencing