Project description:Human induced pluripotent stem cell-derived kidney organoids have potential for disease modelling and regenerative medicine purposes. However, they lack a functional vasculature and remain immature in in vitro culture. Here, we transplanted kidney organoids at day 7+12 of differentiation in the coelomic cavity of chicken embryos and then compared them to their respective untransplanted controls at d7+13 and d7+20 using scRNAseq and imaging modalities. We demonstrate vascularization and enhanced maturation of transplanted kidney organoids.
Project description:Single cell RNA-seq samples from 3 species across a differentiation from induced pluripotent stem cells to neural progenitor cells were generated to study gene expression evolution. Briefly, previously generated urinary stem cell derived iPSCs of 3 human (Homo sapiens) individuals (3 clones), 1 gorilla (Gorilla gorilla) individual (2 clones) and fibroblast derived cynomolgus macaque (Macaca fascicularis) iPSCs of 2 individuals (4 clones) (Geuder et al. 2021) were differentiated to neural progenitor cells via dual-SMAD inhibition as three-dimensional aggregation culture (Chambers et al. 2009; Ohnuki et al. 2014). Single cell RNA-seq libraries from differentiation days 0, 1, 3, 5, 7, 9 were generated using mcSCRB-seq protocol (Bagnoli et al. 2018).
Project description:BET-regulated transcriptome and BRD4, BRD2, BRD3 and Pol II ChIP-seq datasets in human ESCs before and after BET inhibition. Transcription factors and chromatin remodeling complexes are key determinants of embryonic stem cell (ESC) identity. In this study, we investigate the role of BRD4, a member of the bromodomain and extra-terminal domain (BET) family of epigenetic reader proteins, in control of ESC identity. We performed RNA-seq analyiss in the presense of small molecule inhibitors of BET proteins to show that BRD4 positively regulates the ESC transcriptome. We also integrated RNA-seq analysis with ChIP-sequencing datasets s for BRD4 (and for other BRD2 and BRD3) to demonstrate that BRD4 binds SEs and regulates the expression of SE-associated pluripotency genes. We have also conducted ChIP-seq analysis for Pol II binding to demonstrate that SE-associated genes depend on BRD4-dependent Pol II binding at TSS and gene body for their productive transcriptional elongation. Total RNA was extracted from samples using the RNeasy Qiagen kit according to the manufacturer’s instructions. Deep sequencing of RNA (1ug) from hESCs FGF- or MS436-treated at day 1 and day 5 was performed as described in (Higgin et al., 2010c). Samples were subjected to PolyA selection using magnetic oligo-dT beads. The resulting RNA samples were then used as input for library construction as described by the manufacturer (Illumina, CA, USA). RNA libraries were then sequenced on the GAIIx system using 50bp single reads. Chromatin for ChIP-sequencing was obtained from FGF-maintained hESCs, vehicle or MS417-treated (at 250nM concentration for 6h) (10 to 20x106 cells/IP). ChIP-Seq libraries were generated using standard Illumina kit and protocol as described in (Ntziachristos et al., 2012). We performed cluster amplification and single read 50 sequencing-method using the Illumina HiSeq 2000, following manufacturer’s protocols.
Project description:RNA-seq samples from 3 species across a differentiation from induced pluripotent stem cells to neural progenitor cells were generated to study gene expression evolution. Briefly, previously generated urinary stem cell derived iPSCs of 3 human (Homo sapiens) individuals (3 clones), 1 gorilla (Gorilla gorilla) individual and fibroblast derived cynomolgus macaque (Macaca fascicularis) iPSCs of 2 individuals (4 clones) (Geuder et al. 2021) were differentiated to neural progenitor cells via dual-SMAD inhibition as three-dimensional aggregation culture (Chambers et al. 2009; Ohnuki et al. 2014). Bulk RNA-seq libraries of iPSCs and NPCs were generated using prime-seq protocol (Janjic et al. 2022).
Project description:Five hiPSC lines from four individuals were differentiated to neural progenitor cells. The two cell types were compared in regards to marker gene expression of known cell type markers to show that the used bulk RNA-seq protocol, called prime-seq can capture known biological differences.
Project description:Here silica column and bead based RNA isolation was performed. The two methods were compared using prime-seq to determine the effect of the RNA isolation method on gene expression quantification. This was tested for three different types of input samples, PBMCs, HEK293T cells and striatal mouse tissue to compare the RNA isolation over a range of inputs. For each sample and experimental condition 8 technical replicates were performed.
Project description:Here a part of the MAQC-III study was repeated with the prime-seq method to have a dataset to compare to this gold standard RNA-seq dataset using power simulations.
Project description:Nine different cell types (common dendritic progenitor (CDP), pre-conventional dendritic cell (pre-cDC), common dendritic progenitor (CDPr, according to Rodrigues et al., but this population was flawed in sorting purity), Flt3+ CD11c+ Siglec-H+ CCR9-low B220-low progenitor cell (lo-lo), Flt3 + CD11c+ Siglec-H+ CCR9-low B220-high progenitor cell (lo-hi), plasmacytoid dendritic cell (pDC), Ly6D+ lymphoid progenitor (SP), Ly6D+ Siglec-H+ lymphoid progenitor (DP) and common lymphoid progenitor (CLP)) were sorted to allow for analysis of their transcriptomic relation and/or similarity. The pDC_precursor_scvelo.h5ad file is a processed file ready for direct downstream analysis with scvelo.
Project description:Here we test the impact of barcode swapping in prime-seq. To this end we isolated RNA from human iPSCs and mouse ESCs, processed them separately using prime-seq but pooled them for cDNA amplification.