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| Status |
Public on Dec 30, 2020 |
| Title |
Terc-/-ESC PDL450times-rep1 |
| Sample type |
SRA |
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| Source name |
mESC
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| Organism |
Mus musculus |
| Characteristics |
cell type: mouse Embryonic Stem Cell
strain: DKO741
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| Treatment protocol |
Cells were longitudinally harvested at 100, 350, 450 and 800 passage
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| Growth protocol |
The Terc targeting construct (TR-10) was transfected into E14 mESC by electroporation to exchange the first Terc allele. The targeting plasmid included PGK-neo gene flanked by loxP sites. The transfected cells were selected with G418 (200 mg/ml) and Ganciclovir (1 mM). The resistant Terc+/- cells were picked after eight days. The neo gene was removed by infection with the adenovirus of Cre recombinase. The Terc+/- cells were re-targeted with TR-10. Two lines of Terc-/- ESC, DKO301 and DKO741 were recovered. Both of DKO301 and DKO741 produced survivors with rare frequencies. We got those ALT survivor cells at each PDL timing (PD100, PD350, PD400, PD450) with control (Terc+/+) cells. For mESC maintenance and stock preparation, mouse embryonic fibroblast (MEF) cells were used as feeder cells. Feeder-free mESCs were used for all experiments including nucleic acids extraction, protein extraction, immunostaining, and fluorescent in situ hybridization.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
50,000~75,000 cells were pelleted at 500 xg, 4°C for 5 min. I added 50 ul of cold RSB (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2) containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin and pipetted 3 times. The cells were incubated on ice for 3min, and washed with 1ml of cold RSB containing 0.1% Tween-20 but no other detergents. The cells were pelleted and resuspended wiht 50 ul of transposition mixture (25 ul 2X TD buffer, 2.5 ul transposase, 16.5 ul PBS, 0.5 ul 1% digitonin, 0.5 ul 10% Tween-20, 5 ul H20) (2X TD buffer=20 mM Tris-Cl, pH 7.6, 10 mM MgCl2, 20% dimethyl formamide). The reaction was incubated at 37°C for 30 min with consistent shaking, and stop the reaction by adding 50 ul of tagmentation stop buffer (10 mM Tris-Cl, pH 8.0, 20 mM EDTA, pH 8.0). The reaction was cleaned with Zymo DNA clean and concentrator-5 kits. DNAs were eluted in 21 ul of elution buffer, and amplified for 5 cycles using NEBnext high-fidelity 2x PCR master mix.
DNA libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
ATAC-seq reads were mapped to the mm10 reference genome using Burrows-Wheeler Aligner (BWA) MEM algorithm (version 0.7.17)
Duplicated reads were marked with Picard-Tools (version 1.96) and Samtools (version 1.9)
Peak-calling was performed using MACS2 (version 2.1.2) callpeak command.
Genome_build: mm10
Supplementary_files_format_and_content: bigwig format of peak
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| Submission date |
Apr 01, 2020 |
| Last update date |
Dec 31, 2020 |
| Contact name |
DAEUN KIM |
| Organization name |
Ajou university
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| Street address |
World cup-ro
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| City |
suwon |
| ZIP/Postal code |
KS002 |
| Country |
South Korea |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE147912 |
Telomeres reforged with non-telomeric sequences in mouse embryonic stem cells (ATAC-seq) |
| GSE147916 |
Telomeres reforged with non-telomeric sequences in mouse embryonic stem cells |
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| Relations |
| BioSample |
SAMN14518707 |
| SRA |
SRX8042361 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4448841_PD450_rep1_ATAC_chr13N.bw |
647.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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