|
| Status |
Public on Apr 05, 2022 |
| Title |
D10_shcontrol_rep2 [RNA-seq] |
| Sample type |
SRA |
| |
|
| Source name |
Reprogramming cells, control shRNA
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Reprogramming cells
day of reprogramming: D10
|
| Treatment protocol |
20,000 MEFs at passage 2 were plated in a 12-well plate and then infected twice with retroviral stocks generated with Plat-E cells. 3 volumes of the supernatants of OSKM transcription factors were mixed with 1 volume of fresh MEF medium containing polybrene at a final concentration of 8 μg/ml. The infection efficiency was tested by MEFs transduced with virus of pMXs-EGFP. pMXs-CTCF and shRNAs were also transfected into OG2-MEFs by retrovirus generated from Plat-E cells. For infecting OG2-MEFs, the medium of OG2-MEFs was replaced with 2 ml of infection mixture per well in a 12-well plate. Infected cells were cultured with mESC medium containing 50 μg/ml Vitamin C (Sigma) post infection and renewed daily. For polycistronic OSKM-mediated reprogramming experiments, MEFs were transduced with 1 volume inducible lentivirus of OSKM and 2 volume rtTA retroviruses generated from HEK293T cells. Infected cells were cultured in mESC medium containing 50 μg/ml Vitamin C and 2 μg/ml doxycycline (Sigma) for 12 days to count GFP positive colonies. To ensure proper CTCF knockdown efficiency before transfecting these virus supernatants into MEFs, we concentrated virus supernatant produced from Ctcf shRNAs.
|
| Growth protocol |
mESCs and iPSCs were cultured in DMEM/High glucose medium (Hyclone) supplemented with beta-mercaptoethanol (10 mM), sodium pyruvate (10 mM), non-essential amino acids (10 mM), GlutaMAX (10 mM), 15% fetal bovine serum (Gibco), 1000 U/ml leukemia inhibitory factor (LIF), 1 μM MEK inhibitor PD0325901 and 3 μM GSK3 inhibitor CH99021. MEF cells were cultured in 10% FBS medium supplemented with non-essential amino acids (10 mM) and GlutaMAX (10 mM).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted with TRIzol.
RNA sequencing libraries were constructed using the NEBNext® Ultra™ RNA Library Prep Kit from Illumina (NEB).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
| |
|
| Description |
rnaseq_shctcf_d10.tsv
|
| Data processing |
RNA-seq raw datasets were qualified with FastQC tool and trimmed with trim galore if containing adaptors.
Reads were aligned to mm10 genome using rsem with options "--star". Counts were extracted from sample.genes.results file and merged together. RNA-seq raw counts were normalized with EDAseq.
Genome_build: mm10 (GRCm38), Ensembl v95
Supplementary_files_format_and_content: tsv files were generated from normalized RNAseq data.
|
| |
|
| Submission date |
Dec 11, 2018 |
| Last update date |
Apr 05, 2022 |
| Contact name |
ya wei song |
| E-mail(s) |
song_yawei@gibh.ac.cn
|
| Phone |
13288824450
|
| Organization name |
Guangzhou Institute of Biomedicine and Health,Chinese Academy of Sciences
|
| Department |
South China Institute for Stem Cell Biology and Regenerative Medicine
|
| Street address |
190 Kai Yuan Avenue, Science Park
|
| City |
Guangzhou |
| State/province |
Guangdong |
| ZIP/Postal code |
510530 |
| Country |
China |
| |
|
| Platform ID |
GPL21273 |
| Series (2) |
| GSE123641 |
CTCF Functions as an Insulator for Somatic Genes and a Structural Regulator for Pluripotent Genes during Reprogramming [RNA-seq] |
| GSE123670 |
CTCF Functions as an Insulator for Somatic Genes and a Structural Regulator for Pluripotent Genes during Reprogramming |
|
| Relations |
| BioSample |
SAMN10581159 |
| SRA |
SRX5125720 |
