|
| Status |
Public on Sep 11, 2019 |
| Title |
WT_ESC_2i_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic Stem Cell
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Embryonic Stem Cell
genotype: Wild type
|
| 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 1g 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 maintained in a pluripotent state through culturing with LIF on gelatinised 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 |
| E-mail(s) |
fitzpadj@tcd.ie
|
| 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 |
|
| Relations |
| BioSample |
SAMN11013765 |
| SRA |
SRX5426141 |
