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| Status |
Public on Dec 08, 2019 |
| Title |
C4_SpenDegron_Dox24h_rep2 |
| Sample type |
SRA |
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| Source name |
ESC_TX1072_SpenAID
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| Organism |
Mus musculus |
| Characteristics |
genotype: Spen-AID
treatment: Dox24h
strain: BL/6J x CAST-EiJ
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| Treatment protocol |
X chromosome inactivation was induced through Xist expression upon administration of doxycycline (1ug/mL). Auxin mediated depletion of target proteins was achieved through supplementing culture media with auxin (Sigma) at the recommended concentration of 500uM. Auxin containing medium was renewed every 24 hours.
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| Growth protocol |
Mouse ES cells (TX1072) were grown on 0.1% gelatin-coated flasks in 8% CO2 37°C incubators. For all experiments, cells were cultured in 2i + LIF serum-containing ES cell medium - DMEM (Sigma), 15% FBS (Gibco), 0.1mM β-mercaptoethanol, 1000 U/ml leukemia inhibitory factor (LIF, Chemicon), CHIR99021 (3uM), PD0325901 (1uM).
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was performed using the RNeasy kit and on-column DNAse digestion (Qiagen). Reverse transcription was performed on 1ug total RNA using Super Script III (Life Technologies). To quantify allelic skewing, cDNA was amplified using biotinylated primers and subsequently sequenced using Q24 Pyromark (Qiagen). Only samples showing a RIN score above 9 were used to prepare RNAseq libraries (TruSeq).
Library were prepared using TruSeq 2 kit.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
All data were mapped to the mouse genome mm10, using the BL6-EiJ / CAST SNPs from the mouse genome project (v5 SNP142), and the gene annotation from ensembl (v92). Analyses were performed in R (v 3.4.2) and Bioconductor (v3.6).
Reads were trimmed using Trimgalore (v 0.4.4), mapped using STAR 10 (2.5.3a, parameters: --outFilterMultimapNmax 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.06 --alignIntronMax 500000 --alignMatesGapMax 500000 --alignEndsType EndToEnd --outSAMattributes NH HI NM MD), and removed when mapping to the mitochondrial genome. Remaining reads were split by allele using SNPsplit (v 0.3.2). Allele specific and the unassigned bam files were sorted, duplicates removed using picard (v2.18.2, parameters: REMOVE_DUPLICATES=true ASSUME_SORTED=true) and pooled as the total reads. Quantification of expression was performed using featureCount (parameters: -p -t exon -g gene_id, -s 1 for stranded RNAseq of in vitro cell, -s 0 for non-stranded RNAseq of single embryo). Data were then analysed in R using DESeq2 13 (v1.18.1), calculating the sizeFactor on the count of total reads and applying it to the allele specific counts.
Genome_build: mm10
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| Submission date |
Jun 11, 2019 |
| Last update date |
Dec 09, 2019 |
| Contact name |
samuel Collombet |
| E-mail(s) |
[email protected]
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| Organization name |
Ecole Normale Superieure
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| Department |
Biology
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| Lab |
Thieffry
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| Street address |
46 rue d'Ulm
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| City |
Paris |
| ZIP/Postal code |
75005 |
| Country |
France |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE131784 |
Spen regulates X chromosome inactivation |
| GSE132560 |
Spen regulates X chromosome inactivation (RNAseq in ESCs ) |
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| Relations |
| BioSample |
SAMN12023335 |
| SRA |
SRX6044201 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3874695_C4_SpenDegron_Dox24h_RNAseq_rep2.bw |
79.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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