Single-cell RNA sequencing of germ-free zebrafish embryos
Ontology highlight
ABSTRACT: The present study was conducted in the frame of the EU-funded Graphene Flagship project. We previously evaluated the impact of graphene oxide (GO) on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild-type versus AhR-deficient zebrafish. Here, we performed single-cell RNA-sequencing (10x Genomics) on whole (dissociated) germ-free (GF) zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.
Project description:The present study was conducted in the frame of the EU-funded Graphene Flagship project. The aim is to evaluate the impact of graphene oxide (GO) on the (innate) immune system using zebrafish as a model. We previously performed single-cell RNA-sequencing of germ-free zebrafish embryos exposed to GO plus the microbial metabolite butyrate (BA). Here, we performed a follow up experiment using germ-free lck-GFP transgenic fish in which the zebrafish were exposed to GO plus BA at 5 dpf. The embryos were then dissociated and subsequently sorted on lck and submitted for single-cell RNA-sequencing using 10x Genomics.
Project description:The spinal cord neural stem cell potential is contained within the ependymal cells lining the central canal. This neural stem cell potential is known to decline with age in the mouse. Here, we microdissected and dissociated into single cells the central canal region from the spinal cord of 4 young adult (3-to-4-month old) and 4 aged (18-to-19-month old) C57BL/6J mice to profile the transcriptomes of cells in and around the central canal using 10x Genomics technology.
Project description:Transcriptome data from zebrafish single cells from guts from either from Tg(lck:EGFP) rag1-/-mutant or wild-type zebrafish were isolated and single cell suspensions were prepared as described in protocol section. Three zebrafish, per each condition (i.e. zebrafish intraperitoneally injected with PBS, lyophilised Anisakis simplex or inactivated Vibrio anguillarum), were used to collect the total of 12,000 lck+ cells (4000 per zebrafish) for 10x experiment.
Project description:Haematopoietic stem cells reside in the bone marrow where they generate the effector cells that drive immune responses. However, in response to inflammation some haematopoietic stem and progenitor cells (HSPC) are recruited to tissue sites and undergo extramedullary haematopoiesis. Contrasting this paradigm here we show, with single cell sequencing, residence and differentiation of HSPC in healthy gingiva, a key oral barrier, in the absence of overt inflammation.
Project description:Differentiation of HES3 hESCs using BMP4, Activin A and CHIR99021. 5 timepoints of sample collection (day 3-12). Differentiations were started sequentially and collected and processed on the same day. Sample1 = day 3 + day3.75 (50:50 mix); Sample2 = day 4.75; Sample3 = day 5.75; Sample 4 = day 12.
Project description:Three cell lines were derived from the A375 human melanoma (harbouring the BRAF v600e mutation; ATCC) after treatment with vemurafenib during a period of 4-5 weeks in the absence (MAPKi-resist Rep1/Rep2) or presence of the fatty acid oxidation inhibitors etomoxir (Etomoxir Rep1/Rep2) or ranolazine (Ranolazine Rep1/Rep2).
Project description:Human embryonic stem cell (hESC) line Man-13 was edited by CRISPR resulting in a hESC line carrying a heterozygous frameshift mutation with a premature stop codon in exon-1 of the HNF1B gene ([p.Val61Argfs*18]), functionally equivalent to a heterozygous whole-gene deletion. Kidney organoids were then created by differentiation (as per Takasato et al, 2015; Bantounas et al, 2018; Bantounas et al 2021) of this line and an isogenic non-mutant control line. Single-cell RNAseq (scRNAseq) was then performed on day-25 of differentiation to identify transcriptomic differences, as well as differences in identity and numbers of the cell populations comprising the organoids, with the aim of mechanistically explaining developmental aberrations observed in patients with mutations in this gene.
Project description:The project aims at linking functionality to metabolic fluxes of mouse primary B cells, potentially correlating antibody secretion rates with metabolic differences in subpopulations of B cells. In previous experiments, subpopulations of primary B cells with different phenotypes were observed. Therefore, the next step is to further characterize these subpopulations of B cells via single-cell RNA sequencing so that we gain insight about cell characteristics and individual gene markers, and their potential biological role. Our hypothesis is that these cells present different expression profiles of key genes and/or are relevant in different immune-related pathways.
Project description:TEC progenitors that express beta 5-t contribute to both the cortical and medullary TEC compartments. Our initial experiments across ageing thymi identified a population of potential progenitor TECs which expanded with age, and appears to be a progenitor population for mTEC. These lineage tracing experiments are designed to chart the altered differentiation and senescence of mTEC progenitors with age.
Project description:The aim was to assess the role of HAND1 in cardiac differentiation. The differentiation protocol was designed to promote cell diversity. For wild-type and HAND1-null cell lines we combined cells differentiating under different conditions: 50% control, 25% SB-treated (day 2-3) and 25% DMH1-treated (day 2-3). The addition of SB at day 2, inhibited SMAD2/3 phosphorylation and created a high BMP signalling bias to restrict cardiac differentiation in favour of other mesodermal lineages, whereas the addition of DMH1 at day 2, inhibited SMAD1/5/8 phosphorylation to create a high Activin signaling bias to promote the co-differentiation of endoderm. Samples were collected at days 3, 4, 5, 6, 7, 8 and 10 for wild-type and HAND1-null populations. An additional day 7 and 8 sample was included for the wild-type, which was generated in the same condition.