Project description:We report the construction of 5 yeast meiotic cDNA libraries and perform proof-of-principle screens to show that these yeast cDNA libraries can be used to identify genes and gene isoforms that are important for competitive fitness. Samples 1-25 are from different stages of cDNA library construction and deep sequencing was used to characterize gene representation in each yeast cDNA library. Samples 26-175 are from proof-of-principle competitive fitness screens.
Project description:Despite a considerable literature concerning the molecular pathogenesis of pituitary tumors, the mechanisms of pituitary tumors development and progression remain unknown. Four SAGE cDNA libraries were constructed using a pool of mRNA obtained from five GH-, two ACTH-secreting, and four non secreting pituitary tumors (NS), and three normal pituitaries from patients who had accidental death, using I-SAGE kit (Invitrogen). The aim of this study was to evaluate the differential gene expression profile by SAGE genes in different subtypes of pituitary tumors to contribute for understanding of pituitary tumorigenesis.
Project description:To establish a platform for genome-wide targeted proteomics, we synthesized 18040 recombinant proteins (in vitro proteome) from human full-length cDNA libraries. Obtained proteins were digested with trypsin and subjected to LC-MS/MS analysis.
Project description:Despite a considerable literature concerning the molecular pathogenesis of pituitary tumors, the mechanisms of pituitary tumors development and progression remain unknown. Four SAGE cDNA libraries were constructed using a pool of mRNA obtained from five GH-, two ACTH-secreting, and four non secreting pituitary tumors (NS), and three normal pituitaries from patients who had accidental death, using I-SAGE kit (Invitrogen). The aim of this study was to evaluate the differential gene expression profile by SAGE genes in different subtypes of pituitary tumors to contribute for understanding of pituitary tumorigenesis. Comparative analysis of gene expression profiles in subtypes of pituitary tumores.
Project description:BackgroundExogenous cDNA introduced into an experimental system, either intentionally or accidentally, can appear as added read coverage over that gene in next-generation sequencing libraries derived from this system. If not properly recognized and managed, this cross-contamination with exogenous signal can lead to incorrect interpretation of research results. Yet, this problem is not routinely addressed in current sequence processing pipelines.ResultsWe present cDNA-detector, a computational tool to identify and remove exogenous cDNA contamination in DNA sequencing experiments. We demonstrate that cDNA-detector can identify cDNAs quickly and accurately from alignment files. A source inference step attempts to separate endogenous cDNAs (retrocopied genes) from potential cloned, exogenous cDNAs. cDNA-detector provides a mechanism to decontaminate the alignment from detected cDNAs. Simulation studies show that cDNA-detector is highly sensitive and specific, outperforming existing tools. We apply cDNA-detector to several highly-cited public databases (TCGA, ENCODE, NCBI SRA) and show that contaminant genes appear in sequencing experiments where they lead to incorrect coverage peak calls.ConclusionscDNA-detector is a user-friendly and accurate tool to detect and remove cDNA detection in NGS libraries. This two-step design reduces the risk of true variant removal since it allows for manual review of candidates. We find that contamination with intentionally and accidentally introduced cDNAs is an underappreciated problem even in widely-used consortium datasets, where it can lead to spurious results. Our findings highlight the importance of sensitive detection and removal of contaminant cDNA from NGS libraries before downstream analysis.
Project description:To study the biodistribution of DNA origami in zebrafish and to determine differences in biodistribution based on the coating of the nanostructure using oligolysine-PEG copolymers, zebrafish embryos (2dpf) were injected with coated or uncoated DNA origami (wireframe nanosheets) without any targeting molecule, but containing a detection fluorophores. After treatment, the embryos were disassociated into a single-cell suspension and sorted through FACS for embryos containing nanostructures (fluorophores). 10X sc-libraries were generated from the sorted embryos and sequenced to establish cell type predictions for cells with coated and uncoated structures respectively.
Project description:Single-cell RNA sequencing analysis has recently provided snapshots of gene expression of specific cell types and enabled cell types classification within an heterogenous population. As well as transcriptional changes, alternative splicing events and modifications of components of splicing machinery actively contributes in shaping cellular phenotype as well as ageing process and diseases occurence. Current high-throughput single-cell RNA sequencing methods may lack information on cell-specific isoform expression, missing key aspects of cell biology. In the present work we introduce a novel approach using the 10X Genomics Chromium to generate short-read (Illumina) and long-read (Pacific Biosciences Sequel II) RNA-sequencing libraries from the same single cells. This approach produced single cell parallel transcriptional and splicing profiling that demonstrates for the first time cell-type specific isoform expression and alterations at transcriptional levels associated with ageing in haematopoietic stem and progenitor cells