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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 20, 2021 |
| Title |
day 0 oe rep2 |
| Sample type |
SRA |
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| Source name |
MEFs_KDM6A_day0
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| Organism |
Mus musculus |
| Characteristics |
strain: 11004
cell type: Mouse embryonic fibroblasts (MEFs)
genotype/variation: KDM6A overexpression
time: 2 days post infection
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| Growth protocol |
Mouse embryonic fibroblasts (MEFs) were obtained from 13.5-dpc embryos collected from homozygous 4F2A females or C57BL/6 females which had been mated with DBA/2 males. 293T and MEFs were cultured in Dulbecco's modified eagle medium (DMEM) containing 15%(vol/vol) fetal bovine serum (FBS), 1X MEM NEAA, 1X L-Glutamine and 1X Penicillin-Streptomycin. (FBS from BI and others from Invitrogen). For iPSC reprogramming, infected or drug treated 4F2A MEFs were cultured in KnockOut DMEM with 15% KnockOut Serum Replacement, 1X MEM NEAA, 1X L-Glutamine, 1X Penicillin-Streptomycin, 1X Sodium Pyruvate, 1000U/ml LIF, 1X 2-Mercaptoethanol and 2 μg/ml doxycycline (LIF from Millipore, doxycycline from Sigma-Aldrich and others from Invitrogen), the medium were changed every day.
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| Extracted molecule |
total RNA |
| Extraction protocol |
48 hrs after infection or 72h post DOX was added, the total RNA of 4F2A MEFs was harvested by using TRIzol reagent (Invitrogen).Total RNA was extracted according to manual instruction. About 60 mg of tissues were ground into powder by liquid nitrogen in a 2 mL tube, followed by being homogenized for 2 minutes and rested horizontally for 5 minutes. The mix was centrifuged for 5 minutes at 12,000×g at 4°C, then the supernatant was transferred into a new EP tube with 0.3 mL chloroform/isoamyl alcohol (24:1). The mix was shacked vigorously for 15s, and then centrifuged at 12,000×g for 10 minutes at 4°C. After centrifugation, the upper aqueous phase where RNA remained was transferred into a new tube with equal volume of supernatant of isopropyl alcohol, then centrifuged at 13,600 rpm for 20 minutes at 4°C. After deserting the supernatant, the RNA pellet was washed twice with 1 mL 75% ethanol, then the mix was centrifuged at 13,600 rpm for 3 minutes at 4°C to collect residual ethanol, followed by the pellet air dry for 5-10 minutes in the biosafety cabinet. Finally, 25μL~100μL of DEPC-treated water was added to dissolve the RNA. Subsequently, total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA).
Oligo(dT)-attached magnetic beads were used to purified mRNA. Purified mRNA was fragmented into small pieces with fragment buffer at appropriate temperature. Then First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products from previous step were heated denatured and circularized by the splint oligo sequence to get the final library. The single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated on BGIseq500 platform (BGI-Shenzhen, China).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
BGISEQ-500 |
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| Description |
exp_D0_KDM6A_2
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| Data processing |
HISAT v2.0.4 --phred64 --sensitive --no-discordant --no-mixed -I 1 -X 1000
Genome_build: GCF_000001635.26_GRCm38.p6
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample
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| Submission date |
Oct 14, 2019 |
| Last update date |
Oct 20, 2021 |
| Contact name |
Qi Jiang |
| E-mail(s) |
[email protected]
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| Organization name |
Harbin Medical University
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| Department |
Department of histology and embryology
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| Street address |
baojian road 157
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| City |
harbin |
| State/province |
heilongjiang |
| ZIP/Postal code |
150081 |
| Country |
China |
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| Platform ID |
GPL23479 |
| Series (2) |
| GSE138815 |
Histone demethylase KDM6A promotes somatic cell reprogramming by epigenetically regulate PTEN and IL-6 signal pathway [RNA-seq] |
| GSE138817 |
Histone demethylase KDM6A promotes somatic cell reprogramming by epigenetically regulate PTEN and IL-6 signal pathway |
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| Relations |
| BioSample |
SAMN13025391 |
| SRA |
SRX6986727 |
| 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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