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A Universal 6iL/E4 Culture System for Deriving and Maintaining Embryonic Stem Cells Across Mammalian Species

ABSTRACT: The derivation of authentic embryonic stem cells (ESCs) across mammalian species remains a major challenge. Here, we report the development of a defined, serum-free culture system, termed 6iL/E4, that enables the derivation and long-term self-renewal of ESCs across diverse mammalian species. To further characterize the cellular states, we performed single-cell RNA sequencing (scRNA-seq) on 6iL-hiPSCs and 6iL-mESCs. Library preparation was carried out using the Single Cell 3′ RNA Prep T2 kit (Illumina). For indexing, the Mix 7 index set was used for 6iL-hiPSC samples and the Mix 8 index set for 6iL-mESC samples. Sequencing was performed on a NovaSeq X Plus platform using paired-end 150 bp reads (2 × 150 bp), generating approximately 200 million reads per sample (100 million reads per direction).

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE325170 | GEO | 2026/06/02

REPOSITORIES: GEO

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A Universal 6iL/E4 Culture System for Deriving and Maintaining Embryonic Stem Cells Across Mammalian Species [EM-seq]

Project description:The derivation of authentic embryonic stem cells (ESCs) across mammalian species remains a major challenge. Here, we report the development of a defined, serum-free culture system, termed 6iL/E4, that enables the derivation and long-term self-renewal of ESCs across diverse mammalian species. To further characterize the state of 6iL-hiPSCs, we performed whole-genome bisulfite sequencing (WGBS) to compare 6iL-hiPSCs with hESCs cultured under mTeSR1 and t2iL/Go conditions. Library PCR amplification was performed for five cycles using NEBNext Multiplex Oligos for Enzymatic Methyl-seq. Equimolar amounts of each library were then pooled and quantified by qPCR before paired-end 150-bp sequencing on a 10B 300-cycle shared flow cell using the NovaSeq X Plus platform at the Advanced Genomics Core.

2026-06-02 | GSE333356 | GEO

A Universal 6iL/E4 Culture System for Deriving and Maintaining Embryonic Stem Cells Across Mammalian Species

Project description:Establishing authentic embryonic stem cells (ESCs) from diverse mammalian species is essential for advancing regenerative medicine, developmental biology, and species conservation. Here, we report the development of a robust, serum-free culture system, termed 6iL/E4 that enables the derivation and long-term self-renewal of ESCs from multiple mammalian species, including mouse, rat, bovine, rabbit, and human. Using systematic signaling pathway analysis, we identified key regulators—including STAT3, GSK3α, PDGF, RAF and LATS—critical for ESC maintenance across species. Additionally, inducible expression of KLF2 and NANOG enhances the naïve pluripotency and chimeric potential of bovine ESCs. The E4 basal medium also supports stable ESC growth while minimizing lineage bias. These findings support the evolutionary conservation of pluripotency signaling and provide a universal platform for cross-species stem cell research, disease modeling, and biotechnology applications.

2026-05-18 | GSE295390 | GEO

Identification of the O-GlcNAc-regulated alternative splicing events by performing RNA sequencing on HeLa cells with altered O-GlcNAc level.

Project description:We performed RNA sequencing on HeLa cells treated with OGT inhibitor (Ac4-5SGlcNAc), DMSO, or OGA inhibitor (Thiamet-G) and obtained about 100 million paired-end reads (150 bp) for each replicate. Then the sequencing data was analyzed by MISO to select the O-GlcNAc-regulated alternative splicing events.

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RNA Sequencing Facilitates Quantitative Analysis of Control and GLUT1-HK2-PFKFB3 Overexpressed Transcriptomics in Mouse Skeletal Muscle

Project description:Purpose: RNA-Seq uses next-generation sequencing to analyze expression across the transcriptome, enabling to detect known or novel features and quantify RNA. The goal of this study is to compare transcriptome profiles between control and GLUT1-HK2-PFKFB3 overexpressed mouse skeletal muscle. Methods: Total RNA was isolated from the gastrocnemius muscle of chow diet-fed M;G and control mice at the age of 3-month-old using RNAiso Plus (Takara Bio). Three independent pooled samples per group were analyzed. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. Fragments Per Kb of exon per Million mapped reads (FPKM) were calculated using featureCounts v1.5.0-p3. Results: We mapped about 6 million sequence reads per sample to the mouse genome and identified 54533 transcripts in the gastrocnemius muscle of control and GLUT1-HK2-PFKFB3-overexpressed mice. RNA-seq data confirmed stable expression of 10 known housekeeping genes, and 7 of these were validated with qRT–PCR. A total 664 transtripts were differentially expressed in wildtype and GLUT1-HK2-PFKFB3 overexpressed skeletal muscle with a cutoff of 2.0-fold and p < 0.05 from RNA sequencing analysis. Altered expression of 15 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method.

2022-09-01 | GSE157257 | GEO

A first-in-class, humanized antibody targeting alternatively spliced tissue factor: preclinical evaluation in an orthotopic model of pancreatic ductal adenocarcinoma

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2020-10-01 | GSE158847 | GEO

Pre and post Classical Swine Fever (CSF) vaccination PBMC transcriptomes of Indian Ghurrah and Landrance pigs

Project description:In this study, we generated the PBMC transcriptome data of 3 Landrace, and 3 indigenous Ghurrah pigs before and 7 days after Classical Swine fever vaccination. The piglets were 8-12 weeks of age. The sequencing was done on Illumina X-Ten platform, generating average of 39 million 150 bp paired end reads for each sample.

2021-10-31 | GSE168047 | GEO

Transcriptomic profiles of Huh7 and HLF hepatocellular carcinoma cells under 2D and 3D culture conditions

Project description:This study investigates transcriptomic changes in human hepatocellular carcinoma (HCC) cell lines (Huh7 and HLF) cultured under different microenvironmental conditions, including 2D monolayer, 3D Inject-Embed, and 3D Mix-Embed cultures. RNA-seq was performed on total RNA using Illumina NovaSeq 6000 with paired-end 150 bp reads. Raw sequencing reads were aligned to the Homo sapiens reference genome (GRCh38/hg38) using STAR, and gene-level quantification was performed using featureCounts. The dataset includes raw FASTQ files and processed count and FPKM matrices for each sample. This resource provides insights into the impact of 3D culture on liver cancer gene expression.

2026-04-22 | GSE298810 | GEO

Transcriptome Sequencing Facilitates Quantitative Analysis of BMDM (bone marrow induced macrophages) from FBXW7f/f and LysM-cre FBXW7f/f Transcriptomes before or after LPS treatment

Project description:Purpose: The goals of this study are to compare genes change between FBXW7f/f and LysM-cre FBXW7f/f BMDM after LPS treatment Methods: BMDM mRNA from 6-8weeks FBXW7f/f (WT) and LysM-cre FBXW7f/f (KO) mice were generated by transcription sequencing, using Illumina. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated.

2020-02-20 | GSE125242 | GEO

ATAC-seq of liver samples in Mettl3 flox/flox and hepatocyte-specific Mettl3 knockout (HKO) mice

Project description:Purpose: The goal of this study is to determine whether hepatic deletion of Mettl3 affects chromatin accessibility. Methods: Liver samples were pooled from livers of Mettl3 HKO and Mettl3 flox/flox mice (n=4 for each group). Nuclei was extracted from liver samples, and the nuclei pellet was resuspended in the Tn5 transposase reaction mix. The transposition reaction was incubated at 37°C for 30 min. Equimolar Adapter1 and Adatper 2 were added after transposition, PCR was then performed to amplify the library. After the PCR reaction, libraries were purified with the AMPure beads and library quality was assessed with Qubit. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufactuer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina NovaSeq 6000 platform and 150 bp paired-end reads were generated. ATAC-seq analysis was performed using a standard protocol. Results: The hepatic chromatin accessibility in Mettl3 flox/flox and HKO mice were characterized.

2021-10-29 | GSE141245 | GEO

Assay for transposase-accessible chromatin-sequencing (ATAC-seq) of liver samples in Wtapflox/flox and hepatocyte-specific Wtap knockout (Wtap-HKO) mice

Project description:Purpose: The goal of this study is to determine whether hepatic deletion of Wtapaffects chromatin accessibility. Methods: Each liver sample was pooled from three Wtap-HKO and Wtapflox/flox mice, respectively. Three independent biological replicates of each group were used for ATAC-seq. Nuclei was extracted from liver samples, and the nuclei pellet was resuspended in the Tn5 transposase reaction mix. The transposition reaction was incubated at 37°C for 30 min. Equimolar Adapter1 and Adatper 2 were added after transposition, PCR was then performed to amplify the library. After the PCR reaction, libraries were purified with the AMPure beads and library quality was assessed with Qubit. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufactuer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina NovaSeq 6000 platform and 150 bp paired-end reads were generated. ATAC-seq analysis was performed using a standard protocol. Results: The hepatic chromatin accessibility in Wtapflox/flox and Wtap-HKO mice were characterized.

2022-06-02 | GSE168945 | GEO

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