|
| Status |
Public on Apr 05, 2022 |
| Title |
shLuci_D12_ATAC-rep2 [ATAC-seq] |
| Sample type |
SRA |
| |
|
| Source name |
Reprogramming cells, control shRNA
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Reprogramming cells
day of reprogramming: D12
|
| 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 |
For ATAC-seq cells, a total of 50,000 cells were washed once with cold PBS and resuspended in 50 μl lysis buffer. The suspension of nuclei was then centrifuged for 10 min at 500g at 4°C, followed by the addition of 50 μl transposition reaction mix (10 μl TD buffer, 5 μl Tn5 transposase and 35 μl nuclease-free H2O). DNA was purified using a MinElute Kit (QIAGEN).
ATAC-seq libraries were first subjected to 5 cycles of pre-amplification. To determine the suitable number of cycles required for the second round of PCR the library was assessed by quantitative PCR as described, and the library was then PCR amplified for the appropriate number of cycles. Libraries were purified with AMPure XP beads.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
shLuci_D12_ATAC-rep2
Mm_atac_shctrl_D12.bed.gz
|
| Data processing |
ATAC-seq raw reads were first assessed with FastQC tool and trimmed with trim galore/cutadapt if they contained adaptors.
Reads were aligned to mm10 genome using bowtie2 with parameters "--very-sensitive --end-to-end --no-unal -X 2000". Aligned reads and high-quality (MAPQ >30) reads were extracted using samtools. Picard tools were used to remove duplicates. Peaks were called using MACS2, normalized tracks were generated during peak calling step with parameters "-B --SPMR".
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: Peak files were generated using MACS2, coupled to redefine_peaks() from glbase3. Bigwig files were generated from normalized bdg files using bedGraphToBigWig tool.
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| |
|
| Submission date |
Dec 16, 2020 |
| Last update date |
Apr 05, 2022 |
| Contact name |
ya wei song |
| E-mail(s) |
[email protected]
|
| 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) |
| GSE123667 |
CTCF Functions as an Insulator for Somatic Genes and a Structural Regulator for Pluripotent Genes during Reprogramming [ATAC-seq] |
| GSE123670 |
CTCF Functions as an Insulator for Somatic Genes and a Structural Regulator for Pluripotent Genes during Reprogramming |
|
| Relations |
| BioSample |
SAMN17099342 |
| SRA |
SRX9695135 |
