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| Status |
Public on Apr 05, 2023 |
| Title |
GM21, Day 0, Rep 2, input |
| Sample type |
SRA |
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| Source name |
GM21
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| Organism |
Homo sapiens |
| Characteristics |
antibody: input chromatin
cell line: GM21
cell type: skin fibroblast
time: Day 0
treatment: H-RAS-G12-V overexpression
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| Treatment protocol |
Chromatin was collected from GM21 fibroblasts retrovirally transduced with an empty vector (referred to as day 0) or H-RASG12V at days 10, 18, 22, and 32 after transduction. A total of 0.8-1 × 107 cells (per time point, from 10 pooled biological replicates) were fixed in 1% formaldehyde for 15 min, quenched in 2 M glycine for an additional 5 min, and pelleted by centrifugation at 2,000 r.p.m., 4 °C for 4 min. Nuclei were extracted in extraction buffer 2 (0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1% Triton X-100 and proteinase inhibitor cocktail) on ice for 10 min, followed by centrifugation at 3,000 × g at 4 °C for 10 min. The supernatant was removed, and nuclei were resuspended in nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, and proteinase inhibitor cocktail). Sonication was performed using a Branson sonicator until the desired average fragment size (100–300 bp) was obtained. Soluble chromatin was obtained by centrifugation at 12,000 r.p.m. for 15 min at 4 °C, and chromatin was diluted tenfold.
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| Growth protocol |
Human somatic GM21 foreskin fibroblasts (Coriell Institute, Camden, NJ) and WI-38 human fetal lung fibroblasts (European Collection of Authenticated Cell Cultures, Porton Down, UK) were cultured in a DMEM medium containing 10% fetal bovine serum (FBS) and 1× penicillin/streptomycin (Corning) at 37 °C in a 2% oxygen atmosphere
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Immunoprecipitation was performed overnight at 4 °C with rotation using 1 × 106 cell equivalents per immunoprecipitation using antibodies (5 µg) against H3K4me1 (Active Motif, Carlsbad, MA; Cat no. 39297) and H3K27ac (Active Motif, Carlsbad, MA; Cat no. 39133). Subsequently, 30 µl of Ultralink Resin (Thermo Fisher Scientific, Waltham, MA) was added and allowed to tumble for 4 h at 4 °C. The resin was pelleted by centrifugation and washed three times in low-salt buffer (150 mM NaCl, 0.1% SDS, 1% Triton X-100, 20 mM EDTA, 20 mM Tris-HCl pH 8.0), one time in high-salt buffer (500 mM NaCl, 0.1% SDS, 1% Triton X-100, 20 mM EDTA, 20 mM Tris-HCl pH 8.0), two times in lithium chloride buffer (250 mM LiCl, 1% IGEPAL CA-630, 15 sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0) and two times in TE buffer (10 mM Tris-HCl, 1 mM EDTA). Washed beads were resuspended in elution buffer (10 mM Tris-Cl pH 8.0, 5 mM EDTA, 300 mM NaCl, 0.5% SDS) treated with RNAse H (30 min, 37 °C) and Proteinase K (2 h, 37 °C), 1 µl glycogen (20 mg/ml; Ambion, Austin, TX) was added and decrosslinked overnight at 65 °C. For histone modifications, DNA was recovered by mixing the decrosslinked supernatant with 2.2× SPRI beads followed by 4 min of incubation at room temperature. The SPRI beads were washed twice in 80% ethanol, allowed to dry, and DNA was eluted in 35 µl of 10 mM Tris-Cl pH 8.0.
Libraries were constructed using an Accel-NGS 2S Plus DNA Library kit (21024; Swift Biosciences, Ann Arbor, MI) and amplified for 9 cycles. Libraries were then resuspended in 20 µl of low EDTA–TE buffer. Libraries were quality controlled in an Agilent Technologies 2100 Bioanalyzer (Applied Biosystems, Santa Clara, CA) and quantified using an Invitrogen Qubit DS DNA HS Assay kit (Q32854) (Thermo Fischer Scientific, Waltham, MA).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
fastq files were quality checked with multiqc
fastq files were trimmed with trimmomatic te moreve adapters and aligned using bowtie 2 with the local option
alignment files were cleaned of optical and PCR duplicates with PicardTools and samtools and blacklisted against artifact regions of the hg19 human genome build.
Enriched regions were identified using macs v.2.2.7.1 (macs2 callpeak --nomodel --shiftsize --shift-control--gsize hs -p 1e-3). The identified peaks were subsequently processed using the irreproducibility discovery rate (IDR) pipeline, generating time point-specific reproducible peak sets at each time point. Reproducible peaks were collapsed into master peaksets per histone modification analyzed.
Signal visualization tracks were generated with deeptools using the RPGC approach to obtain 1X coverage after merging time point replicates into a single bam file.
Assembly: hg19
Supplementary files format and content: BigWig and narrowPeak (except input)
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| Submission date |
Jun 11, 2022 |
| Last update date |
Apr 06, 2023 |
| Contact name |
Ricardo Iván Martínez Zamudio |
| E-mail(s) |
rm1238@rwjms.rutgers.edu
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| Organization name |
Rutgers Biomedical and Health Sciences | Rutgers-Robert Wood Johnson Medical School
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| Department |
Pharmacology
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| Lab |
Martínez Zamudio Lab
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| Street address |
675 Hoes Lane West
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| City |
Piscataway |
| State/province |
New Jersey |
| ZIP/Postal code |
08854 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE205898 |
Escape From Oncogene-Induced Senescence is Controlled by POU2F2 and Memorized by Chromatin Scars [ChIP-seq] |
| GSE206496 |
Escape From Oncogene-Induced Senescence is Controlled by POU2F2 and Memorized by Chromatin Scars |
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| Relations |
| BioSample |
SAMN28986787 |
| SRA |
SRX15680609 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6235103_EV_input_2_dedup_blacklisted_merge.bw |
364.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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