|
| Status |
Public on Jan 22, 2017 |
| Title |
DDX5+/+ mESCs Rep #1 |
| Sample type |
SRA |
| |
|
| Source name |
embryonic stem cells_wildtype
|
| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic stem cells
strain: MPI-II
passages: 13-18
genotype/variation: wild type
|
| Treatment protocol |
Generation of DDX5 Knockout mESCs: Two sets of oligonucleotides were separately cloned into a U6-gRNA vector to create paired sgRNA target sites for SpCas9-nickase in the second exon of Ddx5, encoding the DDX5 protein. CRISPR/Cas9 target sites in Ddx5 (NM_007840.3) were designed by http://zifit.partners.org/ZiFiT/CSquare9Nuclease.aspx. Then these two sets of oligonucleotides were synthesized and inserted into the Bbs I site of the U6-gRNA vector. The sequences of two sets of oligonucleotides were as follow: Ddx5-Target1-F: 5’-CACCGAGACCGCGGCCGGGATCGA-3’, Ddx5-Target1-R: 5’-AAACTCGATCCCGGCCGCGGTCTC-3’, Ddx5-Target2-F: 5’-CACCGGAAAGAAGTTTGGAAATCC-3’, Ddx5-Target2-R: 5’-AAACGGATTTCCAAACTTCTTTCC-3’. To generate a stable DDX5-/- mESC line, 2 × 106 mESCs were transfected with 20 μg U6-gRNA vector and 50 μg U6-cas9 plasmids. Cells were selected with 500 μg/ml of G418 (Sigma) after 24 hr of infection and were maintained in selection for 72 hr. Single clones were separated and were seeded in each well of a 48-well plate. Individual wells were inspected by microscopy to exclude polyclonal cell lines. Monoclonal cell lines were expanded. Then, DNA and protein of monoclonal cell lines were extracted to detect the genotype through DNA sequencing after PCR and Western blot.
|
| Growth protocol |
Wild-type (MPI-II) and DDX5 knockout mESCs were cultured in plates coated with feeder cells in DMEM/High Glucose medium (Hyclone) supplemented with b-mercaptoethanol(10mM), sodium pyruvate(10mM), non-essential amino acids(10mM), GlutaMAX(10mM), 15% fetal bovine serum (Gibco), 1000 U/ml leukemia inhibitory factor (LIF), and 1 μM MEK inhibitor PD0325901 and 3μM GSK3 inhibitor CH99021.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Extracted using TRIzol using the manufacturers instructions
RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Ddx5 wild-type MPI-II embryonic stem cells
|
| Data processing |
rsem v1.2.17/bowtie2 v2.2.0 was used to align to the ensembl transcriptome (mm10, v76)
transcripts with more than 25 sequence tags in 2 or more timepoints were kept for further analysis
mapping tag counts were GC normalised using EDASeq v2.0.0
Genome_build: mm10
Supplementary_files_format_and_content: tab separated text file containing ensembl transcript accession, gene name and normalised abundance estimates for each transcript
|
| |
|
| Submission date |
Nov 08, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Andrew P Hutchins |
| E-mail(s) |
andrewh@sustech.edu.cn
|
| Organization name |
Southern University of Science and Technology
|
| Department |
Bioinformatics
|
| Lab |
Bioinformatics and Genomics
|
| Street address |
1088 Xueyuan Rd
|
| City |
Shenzhen |
| ZIP/Postal code |
518055 |
| Country |
China |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE76824 |
DDX5-deficient affect cell fate decision by regulating lineage specific genes and polycomb repressive complexes [RNA-seq] |
| GSE76825 |
DDX5-deficiency affects cell fate decisions by regulating lineage specific genes and polycomb repressive complexes |
|
| Relations |
| BioSample |
SAMN05992306 |
| SRA |
SRX2336348 |
