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| Status |
Public on Jun 30, 2024 |
| Title |
10A YAP5SA, JUN, rep1 |
| Sample type |
SRA |
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| Source name |
MCF10A
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| Organism |
Homo sapiens |
| Characteristics |
antibody: (CST, #9165)
cell line: MCF10A
cell type: breast epithelial
treatment: YAP5SA epxression
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| Treatment protocol |
MCF10A cells overexpressing YAP 5SA were subjected to CU&RUN.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
CUT and RUN experiments were performed as described previously (Kim et al., Nat. Commun., 2022). Briefly, for each CUT and RUN reaction 200,000 cells were trypsinized, washed, resuspended in 100 µl wash buffer (20 mM HEPES, pH7.5, 150 mM NaCl, 0.5 mM Spermidine) and bound to 10 µl activated BioMag®Plus Concanavalin A magnetic beads (Polysciences) for 10 min at room temperature. The cells were then incubated with antibodies diluted in 100 µl antibody buffer (wash buffer + 0.01% digitonine and 2 mM EDTA) overnight at 4°C. IgG rabbit antibody was used as negative control. After incubation with antibodies, the beads were washed in digitonin wash buffer (wash buffer + 0.01% digitonin) and incubated 1h at 4°C with 1 µg/ml protein A/G Micrococcal Nuclease fusion protein (pA/G MNase). After washing with digitonin wash buffer, beads were rinsed with low salt buffer (20 mM HEPES, pH7.5, 0.5 mM Spermidine, 0.01% digitonine), resuspended in 200 µl incubation buffer (20 mM HEPES, pH7.5, 10 mM CaCl2, 0.01% digitonin) and placed at 0°C to initiate cleavage. After 30 min, reactions were stopped by adding 200 µl STOP buffer (170 mM NaCl, 20 mM EGTA, 0.01% digitonin, 50 µg/ml RNAse A) and the samples were incubated 30 min at 37°C to digest the RNA and release the DNA fragments. The samples were then treated with proteinase K for 1 h at 50°C and the DNA was purified using Phenol/Chloroform/Isoamyl alcohol. After precipitation with glycogen and Ethanol, the DNA pellet was resuspended in 0.1 X TE and used for library preparation.
For DNA library generation, the NEBNext®Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs) was used. Adaptor ligation was performed with 1:25 diluted adaptor and 15 cycles were used for library amplification using dual indices (NEB dual index kit). Paired-end 2x25 bp sequencing was performed.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Adapter removal and quality trimming was performed by Cutadapt. Since the carry-over DNA of pAG-MNase purification from E. coli was used as spike-in control, mapping was performed to hg19 and a human repeat-masked E. coli genome by Bowtie2. Paired-end reads mapped to hg19 with inserts <120 bp were extracted using alignment Sieve (deepTools). A scaling factor was inferred by: scaling factor= (mapped reads < 120 bp to hg19)⁄(mapped reads to E.coli).
The scaling factor was used to generate spike-in normalized bigWig files by bamCoverage (deepTools).
Assembly: hg19
Library strategy: CUT&RUN
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| Submission date |
Jun 27, 2023 |
| Last update date |
Jun 30, 2024 |
| Contact name |
Bjoern von Eyss |
| E-mail(s) |
bjoern.voneyss@leibniz-fli.de
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| Organization name |
Leibniz Institute on Aging
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| Lab |
von Eyss
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| Street address |
Beutenbergstr. 11
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| City |
Jena |
| ZIP/Postal code |
07745 |
| Country |
Germany |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE235963 |
A non-canonical repressor function of JUN restrains YAP activity and suppresses YAP-dependent liver cancer growth [CutRun_in_constitutive_5SA_MCF10A] |
| GSE235968 |
A non-canonical repressor function of JUN restrains YAP activity and suppresses YAP-dependent liver cancer growth |
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| Relations |
| BioSample |
SAMN36002186 |
| SRA |
SRX20793807 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7512424_ID569_57_YK_10A_YAP5SA_Rep1_cJUN_smaller_120bp_spikenormalized.bw |
109.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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