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| Status |
Public on Jul 14, 2021 |
| Title |
ChIP_B4N_797_DMSO_rep1_Nov2020 |
| Sample type |
SRA |
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| Source name |
TC-797 NUT Carcinoma cell line
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| Organism |
Homo sapiens |
| Characteristics |
treatment: DMSO
chip antibody: NUT
replicate: 1
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| Treatment protocol |
TC-797 was treated with DMSO or panobinostat (30 nM) for 4 hours.
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| Growth protocol |
NUT carcinoma (NC) cell line TC-797 cells were maintained as monolayer cultures in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Hyclone, South Logan, UT), 1% Penicillin Streptomycin (SV30010, Hyclone) and 1X GlutaMAX (35050061, Gibco/Thermo Fisher Scientific, Waltham, MA).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
3 x 10^7 cells were used for anti-NUT ChIP and 1 x 10^7 cells were used for anti-H3K27ac ChIP. After the treatment, the cells were trypsinized and crosslinked in 10 ml/1 x 10^7 cells of PBS containing 1% formaldehyde (28908, Thermo Fisher Scientific) for 10 min at room temperature. The formaldehyde solution was quenched by adding 2 M glycine to a final concentration of 125 mM. Crosslinked cells were washed in PBS, and the cell pellet was stored in -80 ºC until use. Chromatin from 1 x 10^7 cells was extracted in 1 ml of buffer I (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP40/IGEPAL CA630, 0.25% Triton X-100, 1x cOmplete, EDTA-free Protease Inhibitor Cocktail (11873580001, Millipore Sigma)) for 10 min on ice. The lysate was centrifuged at 1811 x g for 5 min at 4 ºC, and the supernatant was discarded. The pellet was resuspended in 1 ml per 2 x 10^7 cells of buffer II (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, cOmplete EDTA-free Protease Inhibitor Cocktail (Millipore Sigma)) and incubated at room temperature for 10 min with slow, end-over-end rotation. The samples were centrifuged at 1811 x g for 5 min at 4 ºC, and the supernatant was discarded. The pellet was resuspended in 1 m per 2 x 10^7 cells of buffer III (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% sarkosyl, cOmplete, EDTA-free Protease Inhibitor Cocktail (Millipore Sigma)), and transferred to a milliTube 1ml AFA Fiber (520135, Covaris, Woburn, MA), then sheared by a Covaris E220 Focused Ultrasonicator (Covaris). Sheared chromatin was centrifuged at 15,493 x g for 10 min at 4 ºC, and digested with 20 U of benzonase (Sigma-Aldrich, St. Louis, MO) per1 x 10^7 cells. The immunoprecipitation was performed as described (Shiota et al. 2020).
The library was prepared at Genomics Facility at University of Chicago (Chicago, IL; https://fgf.uchicago.edu/).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Reads were trimmed and verified for quality with Trim Galore! (v.0.6.4_dev, https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/).
Trimmed reads were aligned with STAR (v.2.7.5a) to the human genome (GRCh38 primary assembly) using end-to-end alignment (--runMode alignReads --alignIntronMax 1 --alignEndsType EndToEnd).
Alignment files were restricted to named chromosomes (chr1, chr2, …, chrX, chrY). Duplicate reads were filtered out using Picard Tools MarkDuplicates (v.2.23.8).
MACS2 (v.2.2.7.1) was used to identify enrichment peaks in each sample vs its matched input.
Peaks from replicate samples were combined with bedtools2 mergeBed (-d 300) (v2.23.0) to make a superset of peaks per condition.
Genome_build: GRCh38
Supplementary_files_format_and_content: narrowPeak for enrichment peaks
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| Submission date |
Jul 07, 2021 |
| Last update date |
Jul 15, 2021 |
| Contact name |
Hitoshi Shiota |
| E-mail(s) |
[email protected]
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| Phone |
6175254855
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| Organization name |
Brigham and Women's Hospital
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| Street address |
77 Avenue Louis Pasteur, NRB rm652
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| City |
Boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE179692 |
ChIP-seq analysis of BRD4-NUT and H3K27ac distribution in NUT Carcinoma cell line TC-797 treated with DMSO or Panobinostat |
| GSE179694 |
NUT Carcinoma |
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| Relations |
| BioSample |
SAMN20110689 |
| SRA |
SRX11375858 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5428829_ChIP_DMSO_Rep1_S11_NUT_S14_INPUT_peaks.narrowPeak.gz |
297.0 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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