Single Cell Transcriptome Conservation in Cryopreserved Cells and Tissues
Ontology highlight
ABSTRACT: Sample preservation method that maintains transcripts in viable single cells and so allows to disconnect time and place of sampling from subsequent processing steps.
Project description:The use of single-cell technologies for clinical applications requires disconnecting sampling from downstream processing steps. Early sample preservation can further increase robustness and reproducibility by avoiding artifacts introduced during specimen handling. We present FixNCut, a methodology for the reversible fixation of tissue followed by dissociation that overcomes current limitations. We applied FixNCut to human and mouse tissues to demonstrate the preservation of RNA integrity, sequencing library complexity, and cellular composition, while diminishing stress-related artifacts. Besides single-cell RNA sequencing, FixNCut is compatible with multiple single-cell and spatial technologies, making it a versatile tool for robust and flexible study designs.
Project description:Ballan wrasse (Labrus bergylta) is used as a cleaner fish in Norwegian aquaculture to control sea lice. Thus, knowledge on the digestive physiology and nutrient requirements, as well as the ontogeny of the immune system is important. In this study, two different diets were tested; diet 1 was used as control diet consisting of artemia and rotifers cultivated and enriched in the in-house facility at IMR, Austevoll, Bergen (Norway). Diet 2 consists in plankton which has successfully been used before in cod larvae generating more robust individuals. Sampling was done according to the standard length of the larvae rather than age (dph). Sampling point 0 (BW0) (4 mm), sampling point 2 (BW2) (4,5 mm), sampling point 3 (BW3) (5,7 to 6 mm), sampling 4 (BW4) (7 to 7,5 mm), sampling point 5 (BW5) (10 to 10,5 mm), sampling point 6 (BW6) (16 to 16,5 mm), and sampling point 7 (BW7) (25 to 30 mm). Ontogeny of lymphoid organs and mucosal associated lymphoid tissues in ballan wrasse and the effect of different diets were investigated.
Project description:Non-hematopoietic lymph node stromal cells (LNSCs) regulate lymphocyte trafficking, survival, and function for key roles in host defense, autoimmunity, alloimmunity, and lymphoproliferative disorders. However, study of LNSCs in human diseases is complicated by a dependence on viable lymphoid tissues, which are most often excised prior to establishment of a specific diagnosis. Here, we demonstrate that cryopreservation can be used to bank lymphoid tissue for the study of LNSCs in human disease. Using human tonsils, lymphoid tissue fragments were cryopreserved for subsequent enzymatic digestion and recovery of viable non-hematopoietic cells. Flow cytometry and single-cell transcriptomics identified comparable proportions of LNSC cell types in fresh and cryopreserved tissue. Moreover, cryopreservation had little effect on transcriptional profiles, which showed significant overlap between tonsils and lymph nodes. The presence and spatial distribution of transcriptionally defined cell types was confirmed by in situ analyses. Our broadly applicable approach promises to greatly enable research into the roles of LNSC in human disease.
Project description:Kuepfer2005 - Genome-scale metabolic network
of Saccharomyces cerevisiae (iLL672)
This model is described in the article:
Metabolic functions of
duplicate genes in Saccharomyces cerevisiae.
Kuepfer L, Sauer U, Blank LM.
Genome Res. 2005 Oct; 15(10):
1421-1430
Abstract:
The roles of duplicate genes and their contribution to the
phenomenon of enzyme dispensability are a central issue in
molecular and genome evolution. A comprehensive classification
of the mechanisms that may have led to their preservation,
however, is currently lacking. In a systems biology approach,
we classify here back-up, regulatory, and gene dosage functions
for the 105 duplicate gene families of Saccharomyces cerevisiae
metabolism. The key tool was the reconciled genome-scale
metabolic model iLL672, which was based on the older iFF708.
Computational predictions of all metabolic gene knockouts were
validated with the experimentally determined phenotypes of the
entire singleton yeast library of 4658 mutants under five
environmental conditions. iLL672 correctly identified 96%-98%
and 73%-80% of the viable and lethal singleton phenotypes,
respectively. Functional roles for each duplicate family were
identified by integrating the iLL672-predicted in silico
duplicate knockout phenotypes, genome-scale carbon-flux
distributions, singleton mutant phenotypes, and network
topology analysis. The results provide no evidence for a
particular dominant function that maintains duplicate genes in
the genome. In particular, the back-up function is not favored
by evolutionary selection because duplicates do not occur more
frequently in essential reactions than singleton genes. Instead
of a prevailing role, multigene-encoded enzymes cover different
functions. Thus, at least for metabolism, persistence of the
paralog fraction in the genome can be better explained with an
array of different, often overlapping functional roles.
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Project description:In this study, we designed a space simulation study, named “4 Subjects 180-Day Controlled Ecological Life Support System (CELSS) Integration Experiment”, which took place in Shenzhen, China, from June to December 2016. In this experiment, four subjects (3 males and 1 female) lived for 180 days in an enclosed simulated cabin, and multiple-sampling-point DNA methylation data was collected to conduct the epigenic analysis. Peripheral whole blood cells were extracted from all 4 subjects on the 12 sampling points (Pre45, Pre15, R2, R30, R60, R75, R90, R105, R120, R150, R175 and Post30 mission day during the experiment).