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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 12, 2023 |
| Title |
KO15 (timecourse) - day 3 |
| Sample type |
SRA |
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| Source name |
mESC B6129S6F1 Tet1<tm1Koh>
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| Organism |
Mus musculus |
| Characteristics |
tissue: mESC B6129S6F1 Tet1<tm1Koh>
cell line: KO15
cell type: mESC
genotype: Tet1 KO
treatment: day 3
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| Growth protocol |
Anterior neural progenitor differentiation was performed as previously described (Cruz-Molina et al., 2017; Gouti et al., 2014; Luo et al., 2020). Feeder depleted serum cultured ESCs were plated on gelatin coated plates at 10,000 cells/cm2 (90,000 cells per well in a 6-well plate) in advanced N2B27 defined media compromising a 1:1 mix of 1:1 DMEM/F12 (Invitrogen, 12634-010, Invitrogen) and Neurobasal medium (Invitrogen, 10888-022) supplemented with 0.5x B27 without vitamin A (Invitrogen, 12587-010), 0.5x N2 (Invitrogen, 17502-048), 2 mM L-glutamine (Invitrogen, 25030-024), 40 mg/ml BSA fraction V (Invitrogen, 15260037), 0.1 mM 2-mercaptoethanol (Invitrogen, 31350-010) and 100:100 units:µg/ml penicillin:streptomycin (Sigma-Aldrich, P4333; or Gibco, 15140122). Cells were supplemented with 10 ng/ml bFGF (Cat#100-18C, Peprotech) from day 0 until day 3. At day 3 the medium was changed to medium without bFGF. In some experiments, 5 µM of the Wnt inhibitor XAV939 (Sigma-Aldrich, X3004), 2.5 µM of the TGF-β receptor inhibitor SB431542 (Sigma-Aldrich, S4317), 0.1 µM BMP inhibitor LDN193189 (Sigma-Aldrich, SML0559), 0.3 µM or 3 μM GSK3 β inhibitor CHIR99021 (Axon Medchem BV,1408), 5 or 25 ng/ml of Activin A (Peprotechm 120-14E), or equivalent amount of DMSO (vehicle control) were added from day 2 until day 5. The medium was changed every day during differentiation, except on day 1. At day 4, 1.5x amount of medium was added. The experimental end-point is day 5.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted from cells using Trizol using the manufacturer’s instructions.
RNA sequencing (RNAseq) libraries were prepared from 4 µg of total RNA using the KAPA stranded mRNA-seq kit (Roche, KK8421) according to manufacturer’s specifications. 100 nM KAPA-single index adapters (Roche, KK8700) were added to A-tailed cDNA, and libraries were amplified for 10 cycles. Finally, 1x library cleanup was performed using Agencourt AMPure XP beads (Beckman Coulter, A63881). Library fragment size was assessed using Agilent Bioanalyzer 2100 with the High Sensitivity DNA analysis kit (Agilent, 5067-4626) and concentration was determined using Qubit™ dsDNA HS Assay kit (Invitrogen, Q32854). Each library was diluted to 4 nM and pooled for sequencing on an Illumina Hiseq4000, aiming at 15-20 million SE50 reads per sample (19 million reads on average).
SE50
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
antNPCs_F1Tm1_2_counts.csv
antNPCs_F1Tm1_2_TPM.csv
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| Data processing |
Adapters, polyA/T tails, and bad quality reads (Phred score > 20) were trimmed using Trim Galore! (v0.6.4_dev) with default parameters. Reads were aligned to the transcriptome and quantified using Salmon (v0.14.1) (Patro et al., 2017) with default parameters using GENCODE release 23 of the mouse reference transcriptome sequences and the comprehensive gene annotation. Subsequently, the counts were imported into R (v4.0.2) using tximport (v1.18.0). Differentially expressed genes were defined using DEseq2 (v1.30.0) (Love et al., 2014) and log fold changes corrected using “ashr” method (FDR adjusted p.val < 0.05 & |log2(fold change)| > 1.5) (Stephens, 2017). Temporal differentially expressed genes were determined using xxx with default settings. GO term enrichment was performed using Cluster Profiler (3.18.1), while transcription factor enrichment was calculated using TRRUST website (https://www.grnpedia.org/trrust/) (Han et al., 2018). Lineage marker gene sets were determined by stratifying cell types of the mouse gastrulation reference dataset (Pijuan-Sala et al., 2019) per lineage and calculate markers function of Seurat (Hao et al., 2021). Single cell deconvolution was done with R package SCDC (v1.1.3) (Dong et al., 2021), using the two mixed gastrulation samples from the mouse gastrulation reference dataset as reference. TPM values were calculated using tximport.
Assembly: mm10
Supplementary files format and content: tab separated file with raw counts per sample
Supplementary files format and content: tab separated file with TPM normalized counts per sample
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| Submission date |
Oct 05, 2022 |
| Last update date |
Apr 12, 2023 |
| Contact name |
Bernard Klaas van der Veer |
| E-mail(s) |
ben.vanderveer@kuleuven.be
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| Organization name |
KU Leuven
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| Department |
Department of Development and Regeneration
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| Lab |
Laboratory for Stem Cell and Developmental Epigenetics
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| Street address |
Herestraat 49 - box 804
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| City |
Leuven |
| State/province |
Vlaams-Brabant |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE214828 |
Dual gene activating and repressive functions of TET1 in germ layer lineage bifurcation distinguished by genomic context and dependence on 5-methylcytosine oxidation [RNAseq] |
| GSE214845 |
Dual gene activating and repressive functions of TET1 in germ layer lineage bifurcation distinguished by genomic context and dependence on 5-methylcytosine oxidation |
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| Relations |
| BioSample |
SAMN31158824 |
| SRA |
SRX17802695 |
| 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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