Single cell RNA-seq analysis of Etv2 induced MEFs reprogramming.
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA770421 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR16293141_1.fastq.gz | Fastqsanger.gz | |||
| SRR16293141_2.fastq.gz | Fastqsanger.gz | |||
| SRR16293142_1.fastq.gz | Fastqsanger.gz | |||
| SRR16293142_2.fastq.gz | Fastqsanger.gz | |||
| SRR16293143_1.fastq.gz | Fastqsanger.gz |
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Project description:Etv2-induced MEF reprogramming
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Project description:ATAC-seq analysis of Etv2 induced ES/EB, MEFs,Brg1 KO EBs and Brg1 KD MEFs. Methods: EBs were collected at different time points and disaggregated in 0.25% trypsin at 37C for 3 min incubation with gentle agitation followed by inactivation with culture medium containing 10% FBS. Cells were collected by centrifugation at 500g for 5 minutes, washed once with ice-cold PBS. iHA-Etv2 MEFs were harvested from the culture dishes at different time points by treating the cells with 0.25% trypsin at 37C for 4 min incubation followed by neutralizing and collecting the cells with culture medium containing 10% FBS. ATAC reaction was performed with 50,000 cells as previously described18 using the Tn5 transposase (Illumina) and libraries were created at the University of Minnesota Genome Center. Libraries were then sequenced on a NextSeq Illumina platform (2x50 bp) aiming for 25 million reads per sample.
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Project description:ChIP-seq analysis of Etv2 induced MEF reprogramming and EB differentiation.
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Project description:ChIP-seq analysis of Etv2, Brg1 and H3K27ac post Etv2 induction in MEF and ES/EB. Methods: EB or MEF cells were harvested as described above. ChIP assays for ETV2 and H3K27ac were performed from 2 x 10e6 MEFs or 1 x 10e7 EB cells, respectively. Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temperature and the reaction was quenched by glycine at a final concentration of 0.125 M. For the BRG1 ChIP-seq, cells were first crosslinked in 2 mM disuccinimidyl glutarate (DSG; Life Technologies: Cat. #20593) for 30 min then in 1% formaldehyde for 10 min, quenched with glycine for 5 min. The remainder of the ChIP protocol was performed following the protocol described by Magli et al19. We used antibodies against H3K27ac (Abcam, ab4729), ETV2 (Abcam, ab181847), and BRG1 (Abcam ab110641) for the respective pulldowns. For both of the pulldowns, protein A Dynabeads were added to the ChIP reactions and incubated for 30 minutes at room temperature. Magnetic beads were washed and chromatin was eluted. After crosslinking reversal, RNase A, and proteinase K treatment, ChIP DNA was extracted with the Min-Elute PCR purification kit (Qiagen). ChIP DNA was quantified with Quant-it PicoGreen dsDNA Assay Kit (Life Technologies). Sequencing libraries were prepared using equal amount of ChIP DNA per sample as per instructions of SMARTer® ThruPLEX® DNA-Seq Kit (Takara R400675) and SMARTer® DNA Unique Dual Index Kit - 24U Set A (Takara R400665). Sequencing was performed at the University of Minnesota Genome Center with the The NextSeq 550 high-throughput benchtop sequencer.
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