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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 22, 2020 |
| Title |
MEF #1 (ATAC-seq) |
| Sample type |
SRA |
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| Source name |
MEFs
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| Organism |
Mus musculus |
| Characteristics |
day: 0
transfection: None
overexpression: None
knockout: Wildtype
presumed cell type: mouse embryonic fibroblast
strain: C57BL/6J-OG2
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| Treatment protocol |
Plat-E cells were transfected with pMXs vectors for OSKM to produce virus. Plat-E cells were maintained in DMEM/high glucose containing 10% FBS. MEFs within 3 passages were plated at 4000-5000 per square centimetre and infected with retrovirus. We marked the first infection time point as day0, after 2 rounds of 24h infection, the medium was changed to ESC medium, including Vitamin C.
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| Growth protocol |
MEFs were maintained in DMEM/high glucose (Hyclone) containing 10% fetal bovine serum (FBS, PAA), Glutamax (Invitrogen), nonessential amino acids (Invitrogen), and penicillin/streptomycin (Hyclone). Mouse ESCs and iPSCs were cultured on feeder layer (MEF treated with mitomycin-C) in ESC medium containing DMEM/high glucose (Hyclone) supplemented with 15% FBS (GIBCO), Glutamax, nonessential amino acids, sodium pyruvate, penicillin/streptomycin, beta-mercaptoethanol and leukaemia inhibitory factor (LIF). Reprogramming of MEFs was performed either in mES medium. Vitamin C was purchased from Sigma and used at concentration of 50 µg/ml.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ATAC-seq and analysis. A total of 50,000 cells were washed once with cold PBS and re-suspended in 50 μl lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2, 0.1% IGEPAL CA-630). The suspension was then centrifuged at 500 g for 10 minutes at 4°C, followed by addition of 50 μl transposition reaction mix of TruePrep DNA Library Prep Kit (Vazyme, TD502). Samples were then incubated at 37°C for 30 minutes. Transposition reactions were cleaned up using a MinElute PCR Purification Kit (QIAGEN, 28004). ATAC-seq libraries were subjected to five cycles for pre-amplification and amplified by PCR for an appropriate number of cycles. The amplified libraries were purified with a QIAquick PCR Purification Kit (QIAGEN, 28104). Library concentration was measured using VAHTSTM Library Quantification Kit (Vazyme, NQ101). Libraries were sequenced by Vazyme Co., Ltd, China. All sequencing data were mapped onto the mm10 mouse genome assembly using bowtie2 (–very-sensitive). Low quality mapped reads were removed using samtools (view –q 30) and only unique reads mapping to a single genomic location and strand were kept. We removed mitochondrial sequences using ‘grep –v ‘chrM’.
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 |
Reads were aligned to the mouse mm10 genome using bowtie2 v2.2.0
Peaks were discovered using Dfilter (v1.6)
Genome_build: mm10
Supplementary_files_format_and_content: Processed data files are peak bed files
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| Submission date |
Apr 28, 2020 |
| Last update date |
Jul 22, 2020 |
| Contact name |
Andrew P Hutchins |
| E-mail(s) |
andrewh@sustech.edu.cn
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| Organization name |
Southern University of Science and Technology
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| Department |
Bioinformatics
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| Lab |
Bioinformatics and Genomics
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| Street address |
1088 Xueyuan Rd
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| City |
Shenzhen |
| ZIP/Postal code |
518055 |
| Country |
China |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE75005 |
Role of the histone demethylase Kdm6b/Jmjd3 in somatic cell reprogramming |
| GSE149500 |
Role of the histone demethylase Kdm6b/Jmjd3 in somatic cell reprogramming [ATAC-Seq] |
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| Relations |
| BioSample |
SAMN14754775 |
| SRA |
SRX8189453 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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