|
| Status |
Public on Apr 05, 2022 |
| Title |
D6_shCTCF_SSEA1+ [BL-Hi-C] |
| Sample type |
SRA |
| |
|
| Source name |
Reprogramming cells, CTCF shRNA, SSEA1+ subpopulations
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Reprogramming cells
day of reprogramming: D6
|
| 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 |
genomic DNA |
| Extraction protocol |
The BL-Hi-C libraries were performed as previous described. Briefly, the cells were treated with 1% formaldehyde for 10 min at RT and the crosslink was quenched by adding 2.5 M glycine. Then, the cells were treated with 0.1% SDS FA lysis buffer and 1% SDS FA lysis buffer. After that, the genome was digested with enzyme HaeIII into fragments with blunt-ends, which were treated with adenine and ligated with bridge linker containing biotin for 4 hr at 16°C. The unligated DNA fragments were digested with DNA exonuclease. Next, the cells were treated with SDS and proteinase K to digest proteins, and the DNA was purified using phenol-chloroform extraction with ethanol precipitation. Then, the DNA was fragmented into 300 bp on average by sonication and the biotin-labeled DNA fragments were pulled down with streptavidin coated M280 beads.
The BL-Hi-C libraries were constructed into standard Illumina libraries.
|
| |
|
| Library strategy |
Hi-C |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Raw sequence data of BL-Hi-C were subjected to trimLinker tool from ChIA-PET2 to remove linker sequences with parameters "-m 1 -k 2 -e 1 -l 15 -A ACGCGATATCTTATC -B AGTCAGATAAGATAT". The trimmed reads were processed using HiC-Pro.
The resulting valid pairs were balanced and transformed into hic format using juicer tools.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: NOT PROVIDED
|
| |
|
| Submission date |
Dec 16, 2020 |
| 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 |
GPL24247 |
| Series (2) |
| GSE123653 |
CTCF Functions as an Insulator for Somatic Genes and a Structural Regulator for Pluripotent Genes during Reprogramming [BL-Hi-C] |
| GSE123670 |
CTCF Functions as an Insulator for Somatic Genes and a Structural Regulator for Pluripotent Genes during Reprogramming |
|
| Relations |
| BioSample |
SAMN17099243 |
| SRA |
SRX9695117 |
