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| Status |
Public on Apr 25, 2023 |
| Title |
NCI-H295R, H3K27ac, baseline |
| Sample type |
SRA |
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| Source name |
NCI-H295R
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| Organisms |
Drosophila melanogaster; Homo sapiens |
| Characteristics |
cell line: NCI-H295R
cell type: human adrenocortical carcinoma
chip antibody: H3K27ac (Active Motif 39133)
treatment: DMSO
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| Treatment protocol |
NCI-H295R were plated in standard culture medium in 10 cm dishes at a density of 2.4 million cells per plate. To best account for biological variability, experiment was performed using 24 plates of cells total, where 16 plates were reserved for EZH2i administration at the IC-50 dose (62 μM EPZ-6438) and 8 plates were reserved for vehicle administration (equivalent volume of DMSO, these are considered "baseline" NCI-H295R). 24 hours after plating and daily thereafter, media was changed for media containing IC-50 EZH2i or vehicle. After approximately 96 hours of drug administration (120 hours post-plating), cells were harvested for ChIP-seq.
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| Growth protocol |
NCI-H295R were cultured in DMEM/F12 supplemented with 10% Nu serum, 1% ITS-X, and 1% penicillin/streptomycin in a humidified tissue culture incubator with 5% CO2 at 37°C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were harvested for ChIP-seq with Drosophila melanogaster histone spike-in for all epitopes according to Active Motif's Epigenetic Services ChIP Cell Fixation protocol. Briefly, media was supplemented with 1/10 media volume of freshly prepared Formaldehyde Solution (11% formaldehyde, 0.1 M NaCl, 0.5 M EDTA pH 8.0, 1 M HEPES pH 7.9 in nuclease-free water), and plate was agitated for 15 minutes at room temperature. Fixation was stopped with addition of 1/20 volume of Glycine Solution (2.5 M Glycine, MW 75 in nuclease-free water) and incubating at room temperature for 5 minutes. Cells were then scraped, collected into a conical tube, and pelleted at 800xg at 4°C for 10 minutes. Supernatant was aspirated and each tube of cells was re-suspended in 10 mL chilled PBS-Igepal (0.5% Igepal CA-630 in PBS). Cell were pelleted again, supernatant aspirated, and cells resuspended in 10 mL chilled PBS-Igepal supplemented with 100 μL of 100 mM PMSF in ethanol. Cells were pelleted again, supernatant aspirated, snap frozen on dry ice, stored at -80°C, and shipped on dry ice to Active Motif Services (Carlsbad, CA) for chromatin preparation and ChIP-seq. Chromatin was isolated by addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and DNA sheared to an average length of 300-500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin (pooled from all submitted samples) with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (30 μg) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were immunoprecipitated using the following antibodies: EZH2 (Active Motif, Cat. No. 39901; concentration 8 μL Ab/30 μg chromatin), H3K27me3 (ActiveMotif, Cat. No. 39155; concentration 4 μg Ab/30 μg chromatin), H3K27ac (ActiveMotif, Cat. No. 39133; concentration 4 μg Ab/30 μg chromatin), SF1 (EMD Millipore, Cat. No. 07-618; concentration 5 μg Ab/30 μg chromatin) and β-catenin (Invitrogen, Cat. No. 71-2700; concentration 4 μg Ab/30 μg chromatin). Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.
Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. Steps were performed on an automated system (Apollo 342, Wafergen Biosystems/Takara). After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on Illumina's NextSeq 500 (75 nt reads, single end).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
7_0694_00SUMichigan_Untreated_H3K27Ac_hs-dm
Peak files are formatted as broadPeak
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| Data processing |
We used bowtie2 (Langmead and Salzberg, 2012) to align the reads to the hg38 and dm10 version of the human and fly genomes, respectively. Reads overlapping with blacklisted regions (defined by ENCODE), and reads with a mapping score < 20 were filtered.
BAM files were down-sampled to account for Drosophila spike-in control, enabling quantitative comparison of epitopes accounting for net chromatin recruitment across treatment conditions.
To perform ChIP-seq peak calling, we used SPAN and the JBR browser (Shpynov et al., 2021). These tools allow for empirical parameter adjustment after visual inspection of the peaks to obtain the best possible signal-to-noise ratio (FRIP).
BigWigs files were generated using deepTools 2.0 (Ramírez et al., 2016) with RPGC normalization.
Assembly: hg38/dm10
Supplementary files format and content: BigWig
Supplementary files format and content: broadPeaks - called peaks
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| Submission date |
Jun 01, 2022 |
| Last update date |
Apr 25, 2023 |
| Contact name |
Antonio Marcondes Lerario |
| E-mail(s) |
alerario@umich.edu
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| Phone |
7347737640
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| Organization name |
University of Michigan
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| Department |
MEND
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| Lab |
Hammer Lab
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| Street address |
109 Zina Pitcher Place, 1528 BSRB
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| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109 |
| Country |
USA |
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| Platform ID |
GPL22339 |
| Series (2) |
| GSE205258 |
β-catenin programs a tissue-specific epigenetic vulnerability in aggressive adrenocortical carcinoma (ChIP-seq) |
| GSE205283 |
β-catenin programs a tissue-specific epigenetic vulnerability in aggressive adrenocortical carcinoma |
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| Relations |
| BioSample |
SAMN28812759 |
| SRA |
SRX15556420 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6209256_Untreated_H3K27Ac.rmdup.sort.filtered.ucsc.s0.73.bw |
238.4 Mb |
(ftp)(http) |
BW |
| GSM6209256_Untreated_H3K27Ac_rmdup_sort_filtered_ucsc_s0_73_Pooled_Input_rmdup_sort_filtered_ucsc_200_ff30c_1.0E-6_5.peak.txt.gz |
1.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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