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Sample GSM3625729 Query DataSets for GSM3625729
Status Public on Sep 11, 2019
Title KO_EB_D8_rep1
Sample type SRA
Source name Embryoid Body
Organism Mus musculus
Characteristics cell type: Embryoid Body
genotype: Pcl1-3 TKO
Growth protocol Embryonic stem cells (ESCs) were grown on gelatinised culture dishes in GMEM (sigma) supplemented with 10% ES cell qualified FBS (Millipore), 100 U/mL penicillin (Gibco), 100 U/mL streptomycin (Gibco), 50µM β-mercaptoethanol (sigma), 1:100 Glutamax (Gibco), 1:100 non-essential amino acids (Gibco), 1mM sodium pyruvate (Gibco) and 1:500 homemade leukemia inhibitory factor (LIF). For Embryoid body differentiation, 2-3x106 million ESCs were washed three times with PBS (Lonza) and seeded to non-adherent petri-dishes in ESC media without LIF. The media was changed every 2 days. RNA was harvested after 2, 4 and 8 days.
Extracted molecule total RNA
Extraction protocol RNA was extracted using Rneasy Kit (Qiagen) according to manufacturers instructions.
RNA-Seq libraries were prepared using the NEBNext Ultra RNA Library Pep Kit for Illumina (E7770L), according to manufacturer’s instructions. Prior to starting library preparation, RNA concentrations were measured on a Qubit 3.0 and RIN scores calculated using an Agilent RNA ScreenTape (RIN >8.5). A total 1g of high quality RNA was used for library preparation. Following adaptor ligation, DNA was PCR amplified for 8 cycles. DNA purification was then performed using NEBNext Sample Purification Beads (E7767S). The quality of cDNA libraries was analysed on an Agilent High Sensitivity D1000 Screen Tape. The resulting libraries were then used for cluster generation and sequencing using an Illumina NextSeq 500 (ID: NB501524), with 75bp read length.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Description ESCs that have been induced to differentiate by removal of LIF from culture media and growth on non-adherent plates
Data processing Raw sequencing reads were aligned to the reference transcriptome (mm10) using kallisto and counts per transcripts collapsed to counts per gene using tximport. Bigwigs were generated were generated by aligning to the mouse reference genome to where resulting bam files were converted to bigwig format and scaled to reads per billion.
Genome_build: mm10
Submission date Feb 25, 2019
Last update date Sep 12, 2019
Contact name Darren John Fitzpatrick
Organization name Trinity College Dublin
Department Smurfit Institute of Genetics
Street address College Green
City Dublin 2
State/province Ireland
ZIP/Postal code Eire
Country Ireland
Platform ID GPL19057
Series (2)
GSE127119 Variant PRC2.1 and PRC2.2 co-operate to direct H3K27 methylations in ESCs [RNA-seq]
GSE127121 Variant PRC2.1 and PRC2.2 co-operate to direct H3K27 methylations in ESCs
BioSample SAMN11013758
SRA SRX5426152

Supplementary file Size Download File type/resource 81.7 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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