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BART-Seq: cost-effective massively parallelized targeted sequencing for genomics, transcriptomics, and single cell analysis [Pluripotency]

ABSTRACT: We describe a novel workflow named Barcode Assembly foR Targeted Sequencing, which is a highly sensitive, quantitative, and inexpensive technique for targeted sequencing of transcript cohorts (rBART-Seq) or genomic regions (gBART-Seq) from thousands of bulk samples or single cells in parallel. Multiplexing is based on a simple method that produces extensive matrices of diverse DNA barcodes attached to invariant primer sets, for generating amplicons with dual indices. Here, we used the rBART-Seq for RNA quantification, and for the analysis of developmental states of thousands of single human pluripotent stem cells maintained in different media (mTeSR™1, KSR-bFGF, and E8).

ORGANISM(S): Homo sapiens

PROVIDER: GSE130422 | GEO | 2019/07/07

REPOSITORIES: GEO

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BART-Seq: cost-effective massively parallelized targeted sequencing for genomics, transcriptomics, and single cell analysis [Genotyping]

Project description:We describe a novel workflow named Barcode Assembly foR Targeted Sequencing, which is a highly sensitive, quantitative, and inexpensive technique for targeted sequencing of transcript cohorts (rBART-Seq) or genomic regions (gBART-Seq) from thousands of bulk samples or single cells in parallel. Multiplexing is based on a simple method that produces extensive matrices of diverse DNA barcodes attached to invariant primer sets, for generating amplicons with dual indices. Here, we used the gBART-Seq for genetic screening of breast cancer patients and identified BRCA mutations with very high precision.

2019-07-07 | GSE130421 | GEO

BART-Seq: cost-effective massively parallelized targeted sequencing for genomics, transcriptomics, and single cell analysis [Differentiation]

Project description:We describe a novel workflow named Barcode Assembly foR Targeted Sequencing, which is a highly sensitive, quantitative, and inexpensive technique for targeted sequencing of transcript cohorts (rBART-Seq) or genomic regions (gBART-Seq) from thousands of bulk samples or single cells in parallel. Multiplexing is based on a simple method that produces extensive matrices of diverse DNA barcodes attached to invariant primer sets, for generating amplicons with dual indices. Here, we used the rBART-Seq to analyze cell subpopulations that emerge during 72 hours of Wnt/β-catenin pathway activation of H9 hESCs using recombinant Wnt protein (rWnt3a), a small molecule inhibitor of GSK-3 (CHIR99021), or Dox-inducible constitutively active β-catenin (ΔN90).

2019-07-07 | GSE130420 | GEO

Human embryonic stem cells in AKIT, E8 and KSR/bFGF culture medium

Project description:Analysis of KhES-1 and H9 human ES cells in growth factors-dependent (E8) and -independent (AKIT) medium in feeder-free culture condition and KSR/bFGF medium on a feeder-layer. Results provide insight into genetic stability in different culture media/conditions.

2017-09-08 | GSE103636 | GEO

Homo sapiens

Project description:Human embryonic stem cells in AKIT, E8 and KSR/bFGF culture medium

| PRJNA403828 | ENA

Transcriptome of ESCs, EpiSCs and reverted ES by MM401 (Mll1 inhibitor) #2

Project description:This study describes the transcriptome profiling of: 1) mouse ES cells in LIF/KSR medium; 2)EpiSCs in bFGF/serum-free (KSR) medium; 3) EpiSCs treated with MM401/LIF KSR at D3 and D6 (P2); 4) rES reverted form EpiSC by MM401/LIF KSR treatment at P6, P30 with or without MM401 .

2016-05-21 | GSE73993 | GEO

Simple differentiation method using FBS identifies DUSP6 as a marker for fine-tuning of FGF-ERK signaling activity in human pluripotent stem cells

Project description:Assessment of differentiation potential is a basic requirement to obtain qualified human pluripotent stem cells (hPSCs). Here, we report a simple differentiation method using fetal bovine serum (FBS) to estimate differentiation potential and propensity of hPSCs. PluriTest using RNA-sequencing showed that cells differentiated after treatment with 5% FBS. Expression patterns of three germ layer markers revealed that cells cultured in Knockout Serum Replacement-containing medium (KSR) with mouse feeder cells had higher differentiation potential than cells cultured in a chemically defined medium (E8) with recombinant matrix proteins, especially into the mesoderm and endoderm lineages. Analysis of differentially expressed genes between KSR and E8 identified DUSP6 as a marker for where cells had been cultured. Expression of DUSP6 correlated with FGF-ERK signaling activity. Fine-tuning of FGF-ERK signaling activity to a range that can shut down DUSP6 transcription but sustain NANOG transcription partially increased the differentiation potential. Our data suggest that differentiation with 5% FBS is good to estimate differentiation potential and propensity at the early stage, and that DUSP6 is an excellent marker to monitor ERK signaling activity.

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Primed and naïve marmoset pluripotent stem cells as a model for primate development

Project description:We derived primed pluripotent stem cell cultures (PSCs) from marmoset blastocyst using conventional human PSC culture conditions (KSR/bFGF on feeders). Naïve marmoset PSCs were established by chemical resetting. In addition, we generated forebrain-derived cells from the frontal lobe of a marmoset neonate. Individual cells were manually isolated with a mouth pipette and subjected to single-cell full-lengths transcriptome profiling using a modified version of Smart-Seq2.

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Establishment of Bovine Induced Pluripotent Stem Cells

Project description:Pluripotent stem cells (PSCs) have been successfully developed in many species. However, generating bovine induced pluripotent stem cells (biPSCs) has been challenging. Here we report the generation of biPSCs by overexpression of lysine-specific demethylase 4A (KDM4A) and repro-gramming factors OCT4, SOX2, KLF4, cMYC, LIN28, and NANOG (KdOSKMLN). These biPSCs exhibited silenced transgene expression at passage 10, and had prolonged self-renewal capacity for over 70 passages. The biPSCs have flat, primed-like PSC colony morphology in combined me-dia of knockout serum replacement (KSR) and mTeSR, but switched to dome-shaped, naïve-like PSC colony morphology in mTeSR medium and 2i/LIF with single cell colonization capacity. These cells have comparable proliferation rate to the reported primed- or naïve-state human PSCs, with three-germ layer differentiation capacity and normal karyotype. Transcriptome analysis revealed a high similarity of biPSCs to reported bovine embryonic stem cells (ESCs) and embryos. The naïve-like biPSCs can be incorporated into mouse embryos, with the extended capacity of in-tegration into extra-embryonic tissues. Finally, 24.6% cloning efficiency could be achieved in nu-clear transfer (NT) experiment using late passage biPSCs as nuclear donors. Our report represents a significant advance in the establishment of bovine PSCs.

2021-09-27 | GSE180931 | GEO

Genome-wide analysis of gene expression in human embryonic stem cells (hESCs) and their differentiated cells

Project description:In order to investigate the gene expression changes in human embryonic stem cells (hESCs) during differentiation, we performed a microarray analysis from RNAs isolated from undifferentiated hESCs and their differentiated cells incubated for 1 week or 2 weeks in ESC medium. Human SNUhES3 ESCs (Seoul National University Hospital, Seoul, Korea) were cultured on mitotically-arrested STO feeder cells (ATCC, Manassas, USA) in DMEM/F12 supplemented with 20% knockout serum replacement (KSR). The embryoid bodies were incubated for 1 or 2 weeks in the above ESC medium without bFGF. Cells were then lysed and RNA was isolated.

2012-12-31 | E-GEOD-36255 | biostudies-arrayexpress

Secreted footprint of pluripotency in REBL.PAT IPSCs

Project description:With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFβ deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.

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