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| Status |
Public on Dec 08, 2019 |
| Title |
embryo_spenKO_f4_1 |
| Sample type |
SRA |
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| Source name |
embryo_SPEN_KO
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| Organism |
Mus musculus |
| Characteristics |
genotype: Spen_KO
strain: BL/6J x CAST-EiJ
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| Treatment protocol |
E3.5 embryos were collected and morphologically assessed to ensure only viable samples were collected. Zona pellucida was removed by treatment with acidified Tyrode’s solution. Single embryos were picked into individual tubes and cDNA was prepared and amplified.
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| Growth protocol |
Timed natural matings were used for all experiments. Noon of the day when the vaginal plugs of mated females were identified was scored as E0.5. For Spen matings a published conditional allele was used . For oocyte deletions published Rosa26:Zp3-Cre allele was used 4 . F1 hybrid Spen +/- males were obtained by crossing Spen +/+ CAST/EiJ females with Spen +/- C57BL/6J males. For Spen maternally deleted embryos, Spen flox/flox Zp3-Cre +ve C57BL/6J females were crossed with Spen +/- F1 hybrid males. For Spen control embryos, Spen flox/flox Zp3-Cre -ve C57BL/6J females were crossed with Spen +/- F1 hybrid males. All husbandry and experiments involving mice were carried out in agreement with the guidelines from French legislation and institutional policies.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted according to the manufacturer’s recommendations using RNeasy Mini Kit (Qiagen) with on-column DNAse digestion (Qiagen). Only samples showing a RIN score above 9 were used to prepare RNAseq libraries (TruSeq). Libraries were sequenced using NovaSeq 6000 at PE100 settings.
cDNA were fragmented to ~200 bp with Focused-ultrasonicator (Covaris, Woburn, MA) and adaptor ligated libraries were generated using NEBNext Ultra DNA library Prep Kit for illumina (New England Labs, Ipswich, MA)
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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).
RNAseq data were processed similarly as ChIPseq, except for the mapping when the following parameters were used: --outFilterMultimapNmax 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.06 --alignIntronMax 500000 --alignMatesGapMax 500000 --alignEndsType EndToEnd --outSAMattributes NH HI NM MD. 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 |
| GSE132557 |
Spen regulates X chromosome inactivation (RNAseq in SPEN KO embryos) |
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| Relations |
| BioSample |
SAMN12023283 |
| SRA |
SRX6043599 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3874651_embryo_spenKO_f4_1_RNAseq.Aligned.out.merged.rmdup.bam.bw |
82.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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