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GEO help: Mouse over screen elements for information. |
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Status |
Public on Jan 01, 2019 |
Title |
RNAseq_TX1072_Hdac3KO_24h_Dox_Rep1 |
Sample type |
SRA |
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Source name |
TX1072_Hdac3KO_24h_Dox
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Organism |
Mus musculus |
Characteristics |
strain: CAST / C57BL/6 cell line: TX1072 genotype/variation: Hdac3KO condition: 24h Dox replicate: Rep1
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Treatment protocol |
TX1072 based ESC lines were plated at a density of 1.8mln/T75 flask. After 24hrs of culture in DMEM/15%FCS+LIF+2i cell the medium was supplemented with doxycycline (1ug/ml). Cells were then collected at regular intervals.
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Growth protocol |
TX1072 (mouse, female, [Mus musculus castaneus X C57BL/6] embryonic stem cells) cells have been previously derived in the lab (Schulz et al, 2014). Hdac3KO clones have been derived from the TX1072 line by CRISPR/Cas9 targeting. Cells were cultured on gelatin-coated plates in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 15% foetal bovine serum, 2-mercaptoethanol (0.1mM), LIF(1000u/mL) and 2i (PD0325901 [0.4 mM], CHIR99021 [3 mM]) (Ying et al., 2008).
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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). For cDNA synthesis 1.1ug RNA was reverse transcribed using Superscript III Reverse Transcriptase (Thermo Fisher Scientific). RNA was prepared as described above and quality of samples were verified by Tapestation. Only samples with RIN score above 9 were processed. 100 ng of RNA was used for library preparation using Ovation® Mouse RNAseq System that uses mouse-specific InDA-C depletion of rRNA. Manufacturers recommendations were followed. Libraries were sequenced using HiSeq2500 at PE100 settings.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Description |
A657T3
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Data processing |
Reads were first mapped on rRNA with Tophat (2.1.0) (Trapnell et al., 2009), with options [-g 1 KOno-coverage-search KOlibrary-type fr-secondstrand]. Paired unmapped reads were then used to reconstruct fastq files with bedTools bamToFastq (2.25.0) (Quinlan and Hall, 2010). Those files were then mapped with Tophat (2.1.0) (Trapnell et al., 2009), with options [-p 8 -g 1 -x 1 -N 3 KOread-edit-dist 3 KOno-coverage-search KOlibrary-type fr-secondstrand], with refFlat annotation. Reads covering exons of each gene were then counted with featureCounts (1.5.1) with options [-C -p -s 1 -T 8] (Liao et al., 2014). D-scores were then calculated for genes with minimum 10 allelic reads. Genome_build: mm10 Supplementary_files_format_and_content: Count table per gene including : BL6 counts, CAST counts, d-score, total counts
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Submission date |
Jul 01, 2018 |
Last update date |
Jan 01, 2019 |
Contact name |
Aurélie Bousard |
E-mail(s) |
aurelie.bousard@curie.fr
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Organization name |
Institut Curie
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Street address |
26, rue d'Ulm
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City |
Paris |
ZIP/Postal code |
75005 |
Country |
France |
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Platform ID |
GPL17021 |
Series (2) |
GSE116478 |
RNAseq TX1072 WT and HDAC3KO |
GSE116480 |
The role of early chromatin changes in X chromosome inactivation |
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Relations |
BioSample |
SAMN09531239 |
SRA |
SRX4329477 |
Supplementary file |
Size |
Download |
File type/resource |
GSM3239854_RNAseq_TX1072_Hdac3KO_24h_Dox_Rep1_A657T3_allelicRatio.txt.gz |
213.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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