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| Status |
Public on Sep 15, 2022 |
| Title |
TETG_H3K27ac_rep2_input |
| Sample type |
SRA |
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| Source name |
Mouse Embryonic Fibroblast
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| Organism |
Mus musculus |
| Characteristics |
cell type: Mouse Embryonic Fibroblast
antibody: No antibody
treatment: Infected with TETG virus
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| Treatment protocol |
MEF were infected with viruses encoding the reprogramming factors with polybrene(4ug/ml)
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| Growth protocol |
Mice were raised and maintained under standard laboratory conditions.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin were harvested using truChIP® Chromatin Shearing Kit (Covaris, PN 520154) following the manufacturer’s protocol. Briefly, five million cells were cross-linked with Fixing Buffer A at room temperature for 10 minutes. Quenching Buffer E was added to the fixed cells and the cells were incubated at room temperature for an additional 5 minutes. The cells were washed twice with ice-cold PBS. To extract the nuclei, Lysis Buffer B containing 1x protease inhibitors were added to the cells. Incubate the cell on ice for 10 minutes on a rocker at 4 degree. Intact nuclei were collected by centrifugation at 1,700 x g for 5 minutes at 4 degree. The nuclei were washed once with Wash Buffer C and then Shearing Buffer D3. The pelleted nuclei were resuspended in 1ml Shearing Buffer D3 and transfer to milliTUBE (Covaris, PN 520135). The chromatin shearing were performed with E220 Focused-ultrasonicator (Covaris) with 60sec on and 30sec off, 14 cycles. The chromatin solution was clarified by centrifugation at 20,000 g at 4C for 45 minutes and then pre-cleared with Dynabeads protein A (Life Technologies) for 2 hours at 4C. The pre-cleared chromatin sample was incubated with 50 mL of Dynabeads protein A loaded with 5 mg antibody (anti-H3K27ac, ab4729; anti-Mef2c, ab211493; anti-Ascl1, ab74065) overnight at 4C. The beads were washed three times with 0.1% SDS lysis buffer, once with 0.1% SDS lysis buffer/0.35 M NaCl, once with 10 mM Tris-Cl (pH 8)/1 mM EDTA/0.5% NP40/ 0.25% LiCl/0.5% NaDOC, and once with TE buffer (pH8.0). The immunoprecipitated material was eluted from the beads by heating for 45 minutes at 68C in 50 mM Tris-Cl (pH 7.5), 10 mM EDTA, 1% SDS. To reverse the crosslinks, samples were incubated with 1.5 mg/ml Pronase at 42C for 2 hr followed by 67C for 6 hr. The samples were then extracted with MinElute PCR purification Kit (QIAGEN) and eluted in TE buffer.
ChIP DNA were then sent to UNC Chapel Hill’s High-Throughput Sequencing Facility libraries for ChIP-seq library preparation with Thruplex DNA Seq Library Prep kit (Takara).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
TETG_2_peaks.narrowPeak
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| Data processing |
Qualities of de-barcoded raw sequence reads were checked by fastqc.
Reads were aligned to the mm10 reference genome using Bowtie2.
MACS2 were used for peak calling with following parameter -extsize 200 --nolambda -g mm -p 1e-5
Genome_build: mm10 reference
Supplementary_files_format_and_content: 12 txt files containg H3K27ac Peaks of reprogramming and control group for three reprogramming systems in replicates
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| Submission date |
Dec 29, 2021 |
| Last update date |
Sep 15, 2022 |
| Contact name |
LI QIAN |
| E-mail(s) |
li_qian@med.unc.edu
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| Phone |
919 9624982
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| Organization name |
UNC
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| Department |
Department of pathology
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| Lab |
Qian Lab
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| Street address |
111 Mason Farm Road, MBRB
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| City |
Chapel Hill |
| ZIP/Postal code |
27517 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE192727 |
Cross-lineage Potential of Ascl1 Uncovered by Comparing Diverse Reprogramming Regulatomes [ChIP-seq I] |
| GSE192788 |
Cross-lineage Potential of Ascl1 Uncovered by Comparing Diverse Reprogramming Regulatomes |
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| Relations |
| BioSample |
SAMN24492069 |
| SRA |
SRX13548126 |
| 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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