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:Archaeological bone material is increasingly being sampled for various molecular analyses. Minimally invasive sampling protocols should therefore be utilised whenever possible. We compare the proteomic output of 10 Early Neolithic Bos sp. humeri, from two different preservation contexts, of seven sampling protocols, namely retrieving bone chips (with and without demineralisation), acid etching (FASP and chemical neutralisation), eraser, membrane box, and polishing film. We note that minimally invasive sampling methods can extract a substantial amount of protein groups and provide sufficient taxonomic identifications if preservation conditions are favorable. However, if preservation conditions are poorer more invasive sampling techniques should be considered.
Project description:Cell-laden microgels have been used as tissue building blocks to create three-dimensional (3D) tissues and organs. However, traditional assembly methods can not be used to fabricate functional tissue constructs with biomechanical and structural complexity. In this study, we present directed assembly of cell-laden dual-crosslinkable alginate microgels comprised of oxidized and methacrylated alginate (OMA). Cell-laden OMA microgels can be directly assembled into well-defined 3D shapes and structures under low-level ultraviolet light. Stem cell-laden OMA microgels can be successfully cryopreserved for long-term storage and on-demand applications, and the recovered encapsulated cells maintained equivalent viability and functionality to the freshly processed stem cells. Finally, we have successfully demonstrated that cell-laden microgels can be assembled into complicated 3D tissue structures via freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting. This highly innovative bottom-up strategy using FRESH 3D bioprinting of cell-laden OMA microgels, which are cryopreservable, provides a powerful and highly scalable tool for fabrication of customized and biomimetic 3D tissue constructs.
Project description:Vertebrates share the same general body plan and organs, possess related sets of genes, and rely on similar physiological mechanisms, yet show great diversity in morphology, habitat and behavior. Alteration of gene regulation is thought to be a major mechanism in phenotypic variation and evolution, but relatively little is known about the broad patterns of conservation in gene expression in non-mammalian vertebrates.We measured expression of all known and predicted genes across twenty tissues in chicken, frog and pufferfish. By combining the results with human and mouse data and considering only ten common tissues, we have found evidence of conserved expression for more than a third of unique orthologous genes. We find that, on average, transcription factor gene expression is neither more nor less conserved than that of other genes. Strikingly, conservation of expression correlates poorly with the amount of conserved nonexonic sequence, even using a sequence alignment technique that accounts for non-collinearity in conserved elements. Many genes show conserved human/fish expression despite having almost no nonexonic conserved primary sequence.There are clearly strong evolutionary constraints on tissue-specific gene expression. A major challenge will be to understand the precise mechanisms by which many gene expression patterns remain similar despite extensive cis-regulatory restructuring.
Project description:Novel In-insert FFPE proteomics combines single glass insert FFPE tissue processing with spatial quantitative data-independent mass spectrometry (DIA). The method represents a potent protocol for spatial FFPE tissue subsection processing in combination with laser capture microdissection, achieving sufficient MS signal from 50 um size mammary acini FFPE voxels. By eliminating additional robotic platforms, desalting and detergent removal steps prior to injection to the liquid chromatograph, this method minimizes protein losses and keep the analysis cost effective. In-insert proteomics maintains sufficient sensitivity to preserve spatial context within a single FFPE tissue slide. This project aim was to acquire data-dependent data (DDA) on Exploris 480 to demonstrate the sensitivity of the In-insert protocol sensitivity and benchmark it with intersecting protocol published by Weke et al. (2022).
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:The use of Assay for Transposase-Accessible Chromatin (ATAC-seq) to profile chromatin accessibility has surged over the past years, but its applicability to tissues has been very limited. With the intent of preserving nuclear architecture during long-term storage, cryopreserved nuclei preparations from chicken lung were used to optimize ATAC-seq. Sequencing data were compared with existing DNase-seq, ChIP-seq, and RNA-seq data to evaluate library quality, ultimately resulting in a modified ATAC-seq method capable of generating high quality chromatin accessibility data from cryopreserved nuclei preparations. Using this method, nucleosome-free regions (NFR) identified in chicken lung overlapped half of DNase-I hypersensitive sites, coincided with active histone modifications, and specifically marked actively expressed genes. Notably, sequencing only the subnucleosomal fraction dramatically improved signal, while separation of subnucleosomal reads post-sequencing did not improve signal or peak calling. The broader applicability of this modified ATAC-seq technique was tested using cryopreserved nuclei preparations from pig tissues, resulting in NFR that were highly consistent among biological replicates. Furthermore, tissue-specific NFR were enriched for binding motifs of transcription factors related to tissue-specific functions, and marked genes functionally enriched for tissue-specific processes. Overall, these results provide insights into the optimization of ATAC-seq and a platform for profiling open chromatin in animal tissues.