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Sample GSM3791665 Query DataSets for GSM3791665
Status Public on Jul 22, 2020
Title OSKM Day 5 FLAG-JMJD3 overexpression anti-FLAG
Sample type SRA
Source name Mouse MEFs, OSKM day 5
Organism Mus musculus
Characteristics day: 5
transfection: OSKM lentiviral transfection
overexpression: FLAG-JMJD3
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 or iSF1 medium (DMEM/high glucose supplemented with 10% Knockout Serum Replacement, nonessential amino acids, basic fibroblast growth factor (bFGF) and LIF). Vitamin C was purchased from Sigma and used at concentration of 50 µg/ml. Drosophila S2 cells were obtained from Dr. Jianguo He’s lab and cultured in Schneider’s Drosophila medium (Life technologies, 21720-024) supplemented with 10% FBS.
Extracted molecule genomic DNA
Extraction protocol Cells were cross-linked with 1% formaldehyde for 10 min at room temperature, and then add glycine at 0.125M to quench formaldehyde. Cells were rinsed twice with cold PBS, then lysed in ChIP Lysis Buffer A (50mM HEPES-KOH pH7.3; 140mM NaCl; 1mM EDTA pH8.0; 10% glycerol; 0.5% NP-40; 0.25% Triton-100; protease inhibitor cocktail) for 10 min at 4 ℃. Samples were centrifuged at 1,400g for 5min at 4 ℃, discard the supernatant and re-suspend the samples with ChIP Lysis Buffer B (1% SDS; 50mM Tris-HCl pH8.0; 10mM EDTA; protease inhibitor cocktail) for 10min at 4 ℃. Lysates were sonicated into about 150-300 bp fragments using Bioruptor (Diagenode), and centrifuged at 14,500 g at 4 ℃ for 10 minutes to discard insoluble pellets, and the supernatant was diluted ten times with ChIP IP Buffer (0.01% SDS, 1% Triton-100, 2mM EDTA, 50mM Tris-HCl pH8.0, 150mM NaCl, protease inhibitor cocktail), then incubated with specific antibody overnight at 4 ℃, and add protein A/G Dynabeads (Invitrogen) to capture specific immunoprecipitates. Wash the beads 3 times with low salt buffer (0.1% SDS, 1% Triton-100, 2mM EDTA, 20mM Tris-HCl pH8.0, 150mM NaCl), once with high salt buffer (0.1% SDS, 1% Triton-100, 2mM EDTA, 20mM Tris-HCl pH8.0, 500mM NaCl), once with LiCl buffer 0.25M LiCl, 1% NP-40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl pH8.1), and twice with TE buffer (10mM Tris-HCl, 1mM EDTA pH8.0). After washing, add 200ul 10% Chelex-100 and 2ul of Proteinase K (PK,20mg/ml) to incubate in a Mixing Block (BIOER) at 56 ℃ for 30 min, followed by boiling in water for 10 min, then centrifuged at 5,000 rpm for 5 min, and transfer the supernatant to a new tube. Antibodies used were: anti-H3K27me3 (Millipore, 07-449). Drosophila S2 cells were cross-linked in the same way mentioned above, and lysed in ChIP Lysis Buffer A for 10 min, centrifuged, and re-suspended with ChIP Lysis Buffer B, then mixed together with reprogramming mouse cells with the ratio of 1:2. Once Drosophila S2 cells and reprogramming cells were combined, the sample was treated as a single sample throughout the ChIP-seq experiment. After sonication, samples were centrifuged at 13.200 rpm at 4 ℃ for 10 min, the supernatant was diluted twice with ChIP Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, 167 mM NaCl, protease inhibitor cocktail) and dialyzed with Slide-A-Lyzer Dialysis cassette (Thermo Scientific, 66380) to reduce the high level of SDS followed with incubation with antibody and Dynabeads. Washed beads were eluted with fresh Elution Buffer (50 mM Tris-HCl, pH8.0, 10 mM EDTA, 1.0% SDS) at 56 ℃ for 30 minutes. Centrifuge and incubate the supernatant at 65 4 ℃ for 8-16 h to reverse crosslink the immunoprecipitation DNA. After incubated with RNase A and PK, DNA was purified with phenol:chloroform:isoamyl alcohol (P:C:IA).
Standard Illumina protocols
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2000
Data processing Reads were aligned to the mouse mm10 genome using bowtie2 v2.2.0
Peaks were discovered using MACS2 (Klf4)
Genome_build: mm10
Supplementary_files_format_and_content: Processed data files are peak bed files and bigWig tracks
Submission date May 22, 2019
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 GPL13112
Series (2)
GSE74997 Role of the histone demethylase Kdm6b/Jmjd3 in somatic cell reprogramming [ChIP-Seq]
GSE75005 Role of the histone demethylase Kdm6b/Jmjd3 in somatic cell reprogramming
BioSample SAMN11831411
SRA SRX5883889

Supplementary file Size Download File type/resource
GSM3791665_OSKMD5_anti-JMJD3FLAG.bed.gz 99.2 Kb (ftp)(http) BED 219.3 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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