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| Status |
Public on May 24, 2023 |
| Title |
TET1-IP |
| Sample type |
SRA |
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| Source name |
GC-1
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| Organism |
Mus musculus |
| Characteristics |
cell line: GC-1
cell type: male germ cell
antibody: H3K27me3
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| Treatment protocol |
When GC-1 cells were grown at 75% density, replaced with fresh DMEM consisting of all supplements, and after 30 min, Lipofectamine 3000 reagent was mixed with plasmids by incubating them at room temperature for 15 min in proportion to the manufacturer's instructions and adding them to the medium. fresh DMEM containing all supplements was replaced after 12 h, and after 24 h fresh DMEM was replaced with puromycin (1000 ng /ml) to screen TET1,MYC lentiviral vector-stabilized cell clones. single cell clones were isolated after one week of dilution culture and amplified for the next experiments
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| Growth protocol |
It was cultured in DMEM basal medium (319-005-CL, WISENTINC) supplemented with 10% fetal bovine serum (EVERY GREEN) at 37°C in a humidified environment with 5% CO2,
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The tissue/cell was fixed in 1% formaldehyde for 10 min at room temperature by a vacuum pump, after which 0.125 M glycine was added and the mixture was sat for 5 min to terminate the crosslinking reaction. The tissue was then collected and frozen in liquid nitrogen, and grinded by tissue lyser. The grinded powder was treated with lysis buffer and nucleus was collected by centrifuging at 2000g for 5min. Then, nucleus was treated with nucleus lysis buffer and sonicated to fragment chromatin DNA.
The 10% lysis sonicated chromatin was stored and named “input”, and 80% was used in immunoprecipitation reactions with anti-H3K27me3 antibody（#9733, CST）and named “IP”, and 10% was incubated with rabbit IgG (Cell Signaling Technology) as a negative control and named “IgG”，respectively. The DNA of input and IP was extracted by phenol-chloroform method. The high-throughput DNA sequencing libraries were prepared by using VAHTS Universal DNA Library Prep Kit for Illumina V3（Catalog NO. ND607, Vazyme）. The library products corresponding to 200-500 bps were enriched, quantified and finally sequenced on Novaseq 6000 sequencer (Illumina) with PE150 model.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Raw sequencing data was first filtered by Trimmomatic (version 0.36), low-quality reads were discarded and the reads contaminated with adaptor sequences were trimmed.
The clean reads were used for protein binding site analysis. They were mapped to the reference genome of Mus using STAR software (version 2.5.3a) with default parameters.
The RSeQC（version 2.6）was used for reads distribution analysis.
The MACS2 software（Version 2.1.1）was used for peak calling. The bedtools（Version 2.25.0）was used for peaks annotation and peak distribution analysis.
bigWig files were generated using deepTools.
Supplementary files format and content: bigWig
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| Submission date |
May 22, 2023 |
| Last update date |
May 25, 2023 |
| Contact name |
wang jin |
| E-mail(s) |
wj17835684621@126.com
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| Phone |
17835684621
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| Organization name |
Anhui Medical University
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| Department |
School of Basic Medicine
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| Lab |
Human anatomy and histoembryology
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| Street address |
Shushan District
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| City |
Hefei |
| State/province |
Anhui |
| ZIP/Postal code |
230000 |
| Country |
China |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE233150 |
TET1-JMJD3-H3K27me3 targets and regulates Pramel3 to affect spermatogonia self-renewal and proliferation and differentiation |
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| Relations |
| BioSample |
SAMN35309430 |
| SRA |
SRX20465867 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7404117_TET1_IP.bw |
205.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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