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Sample GSM3239854 Query DataSets for GSM3239854
Status Public on Jan 01, 2019
Title RNAseq_TX1072_Hdac3KO_24h_Dox_Rep1
Sample type SRA
 
Source name TX1072_Hdac3KO_24h_Dox
Organism Mus musculus
Characteristics strain: CAST / C57BL/6
cell line: TX1072
genotype/variation: Hdac3KO
condition: 24h Dox
replicate: Rep1
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.
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).
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Description A657T3
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
 
Submission date Jul 01, 2018
Last update date Jan 01, 2019
Contact name Aurélie Bousard
E-mail(s) aurelie.bousard@curie.fr
Organization name Institut Curie
Street address 26, rue d'Ulm
City Paris
ZIP/Postal code 75005
Country France
 
Platform ID GPL17021
Series (2)
GSE116478 RNAseq TX1072 WT and HDAC3KO
GSE116480 The role of early chromatin changes in X chromosome inactivation
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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