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| Status |
Public on Aug 01, 2022 |
| Title |
D0_Naïve_PXGL_scRNAseq |
| Sample type |
SRA |
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| Source name |
Human induced pluripotent stem cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: naive pluripotent stem cells
cell line: ICSIG-1 Sigma hiPSC line
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| Growth protocol |
Naive hPSCs (H9 hESCs and Sigma hiPSCs) were cultured on mitotically inactivated MEF feeders in 5% O2 and 5% CO2 incubator under humidified conditions at 37°C. All naive hPSCs were cultured in PXGL medium (Bredenkamp/Guo Stem cell reports 2019 (PMID: 31708477) which consists of 1:1 DMEM/F12 and Neurobasal, 0.5% N2-supplement, 1% B27-Supplement, 2 mM L-Glutamine, 0.1 mM β-mercaptoethanol, 1x penicillin-streptomycin, 1 μM PD0325901 (Axon Medchem and Wellcome-MRC Cambridge Stem Cell Institute), 2 μM XAV939 (Sigma), 2 μM Gö6983 (Tocris), 20 ng/mL human LIF (PeproTech and Wellcome-MRC Cambridge Stem Cell Institute) and 10 μM Y-27632 (Tocris and Cell Guidance Systems). Naive hPSCs were passaged every 4-5 days in a ratio 1:2 or 1:3 by single-cell dissociation with Accutase followed by filtering through a 40 μm cell strainer.
Human naive to trophoblast and EXMC conversion was done using the following previously described protocol (Dong et al. 2020; Cinkornpumin et al. 2020). Naive hPSCs were seeded in such a way that expecting at least 90% confluency by Day 2 on mitomycin C treated MEFs after dissociating in single-cell using TryplE (for 15 min at 37°C) in their respective naive culture conditions supplemented with 10μM Y-27632. The very next day the media was switched from naive to trophoblast stem cell medium (Okae et al. 2018) consisting of DMEM/F12 (Gibco) supplemented with 0.3% BSA, 0.2% FBS, 1% Penicillin-streptomycin, 1% insulin-transferrin-selenium-ethanolamine-X 100 supplement, 1.5 μg/ml L-ascorbic acid, 0.5 μM A83-01, 1μM SB431542, 50ng/ml hEGF, 2uM CHIR99021, 0.8 mM Valproic acid, 0.1 mM β-mercapto-EtOH (Gibco) and 5 μM Y-27632. The medium was changed every two days supplemented with 5 μM Y-27632. From passage 1 onwards cultures were cultured and maintained on 5 μg/ml Collagen IV coated tissue culture treated plates in hypoxia conditions (5% O2 and 5% CO2) and passaged every 4 days in 1:3 or 1:6 splitting ratios.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were washed with 1X PBS before dissociation from the culture dish by TryplETM Express Enzyme (ThermoFisher, 12605-036) incubation for 8 minutes at 37°C. Single cell suspension was filtered through a Falcon 40 µm Cell Strainer and centrifuged at 200G for 5 min. After resuspension in 1X PBS with 0.04% BSA, the single cell suspension was counted with a Luna-FL automated Fluorescence Cell Counter (Logos Biosystems). TSCs were mixed with primed and naive hPSCs using the following ratio (H9 primed: Sigma primed: H9 naive: Sigma naive: Sigma TSCs = 1:1:1:1:2)
Cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) targeting 2000 cells (Next GEM Single Cell 3’ library and Gel Bead Kit v3.1) according to the manufacturer’s protocol (10x User Guide; CG000204, Revision D). Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT cleanup, complementary DNA amplification and library construction were all performed according to the manufacturer’s protocol. Individual sample quality was assessed using a Tapestation (Agilent). Qubit 2.0 (ThermoFisher Scientific) and KAPA Library Quantification Kit for Illumina Platform (KAPA Biosystems) were used for library quantification before pooling. The final library pool was sequenced on the NovaSeq6000 (Illumina) instrument using NovaSeq SP kit (Illumina) for 2 lanes of 100-base-pair paired-end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10X Genomics scRNAseq
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| Data processing |
Raw sequence reads were quality-checked using the FastQC software. The CellRanger version 4.0.0 was used to process, align and summarize unique molecular identifier (UMI) counts against the 10X Genomics pre-built human GRCh38 and mouse mm10 reference genome dataset (2020-A, July 7, 2020). Downstream analyses were performed on R using Seurat (v4.0.1) (Satija et al. 2015). Human cells were retained and mouse cells (MEFs) were filtered out by adjusting the number of counts per cell (nCount_RNA) and the number of mapped genes per cell (nFeature_RNA) to only keep cells that were mostly mapped to the human GRCh38 (hg38) genome. Cells with more than 25% of mitochondrial counts were filtered out. Count matrix was normalised with Seurat global-scaling normalization method “LogNormalize'' that normalizes the feature expression measurements for each cell by the total expression, multiplies this by a 10000 scale factor, and log-transforms the result.
Assembly: Human GRCh38 (hg38) GENCODE v32/Ensembl 98 and mouse GRCm28 (mm10) GENCODE vM23/Ensembl 98 reference genome dataset (version 2020-A, July 7 2020).
Supplementary files format and content: count matrix includes counts per gene per cell (.csv) and annotations describes (.csv).
Supplementary files format and content: loom files.
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| Submission date |
May 25, 2022 |
| Last update date |
Aug 01, 2022 |
| Contact name |
Vincent Pasque |
| E-mail(s) |
vincent.pasque@kuleuven.be
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| Organization name |
KU Leuven
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| Department |
Development and Regeneration
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| Street address |
Herestraat 49 bus 804
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| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE191286 |
Modeling human extraembryonic mesoderm cells using naive pluripotent stem cells |
| GSE204779 |
Modeling human extraembryonic mesoderm cells using naive pluripotent stem cells [scRNA_seq_timecourse_naiveTSC] |
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| Relations |
| BioSample |
SAMN28657020 |
| SRA |
SRX15449734 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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