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Sample GSM4503991 Query DataSets for GSM4503991
Status Public on Jul 22, 2020
Title OSKMD10 FLAG #1 (ATAC-seq)
Sample type SRA
Source name Mouse MEFs, OSKM day 10
Organism Mus musculus
Characteristics day: 10
transfection: OSKM retroiviral transfection
overexpression: FLAG
knockout: Wildtype
presumed cell type: Intermediate pluripotent reprogrammed cells
strain: C57BL/6J-OG2
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.
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.
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
Library strategy ATAC-seq
Library source genomic
Library selection other
Instrument model Illumina NovaSeq 6000
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
Submission date Apr 28, 2020
Last update date Jul 22, 2020
Contact name Andrew P Hutchins
Organization name Southern University of Science and Technology
Department Bioinformatics
Lab Bioinformatics and Genomics
Street address 1088 Xueyuan Rd
City Shenzhen
ZIP/Postal code 518055
Country China
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]
BioSample SAMN14754774
SRA SRX8189454

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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