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| Status |
Public on Jan 28, 2025 |
| Title |
Control_Tys |
| Sample type |
SRA |
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| Source name |
HPAEC
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HPAEC
cell type: Lung endothelial cells
chip antibody: Histone H3K9 (EMD Millipore #06-942, Temecula, CA)
treatment: Control
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| Treatment protocol |
2 hours prior treatments, cell culture media was changed to 2% FBS EGM-2 and cells were treated with HK-MRSA (2.5 x10^8/ml), Tysiponate (1 μΜ), HK-MRSA +Tysiponate , or PBS (control) for 30 minutes.
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| Growth protocol |
HPAEC were grown in D150 cell culture dishes in EGM-2 media (Lonza, CC-3162) supplemented with 10% FBS at 37°C and 5% CO2 until confluent .
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells from 4 x D150 were pooled for each condition.Step 1 – Cross-Linking and Cell Harvesting: Cells were cross-linked with 1% formaldehyde for 10 minutes to fix protein-DNA interactions. Cross-linking reaction was stopped by adding glycine and cells were harvested, pelleted by centrifugation, and washed with cold PBS. Step 2 – Cell Lysis and Chromatin Isolation: The cell pellets were lysed with ChIP lysis buffer and bulk chromatin was isolated by centrifugation Step 3 – Chromatin Shearing: Isolated chromatin was sheared to 150-300 bp using a cominbination of enzymatic digestion and sonication. Step 4 – Chromatin Pre-Clearing and Antibody Incubation: Sheared chromatin was pre-cleared by incubating with Protein A/G agarose beads to reduce non-specific binding. Pre-cleared chromatin was then incubated with acetylated Histone H3K9 antibody overnight. Step 5 – Immunoprecipitation and Washing: Protein A/G magnetic beads were added to the antibody-bound chromatin fractions to facilitate immunoprecipitation. This incubation was carried out for 2 hours. The antibody-bound chromatin was isolated from bulk chromatin with a magnetic separator. To reduce background signal, the isolated chromatin was washed with a series of buffers containing progressively higher concentrations of salt and detergent. Step 6 – Reverse Cross-Linking and DNA Purification: Samples were treated with RNAse to degrade RNA, Proteinase K to degrade proteins, and heat to reverse the cross-linking between proteins and DNA. Step 7 – DNA Isolation and Storage: DNA was isolated and purified with the Qiagen QIAquick PCR Purification Kit and purified DNA samples were stored at -80°C until ready for sequencing.
Libraries were prepered by University of Chicago Core facility using Immumina Library preparation kit and sequenced on the Illumina HiSeq 4000.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Raw reads were aligned to reference genome hg38 using BWA MEM (Li, 2013). Apparent PCR duplicates were removed using Picard MarkDuplicates (Wysoker et al., 2013). Peaks were called against the input sample using Macs2 (Zhang et al., 2008, p. 2). Peaks with a score >5 (-log10 q-value) were retained. For quantitative comparisons, peaks were merged using bedtools merge (Quinlan and Hall, 2010). Enrichment levels for merged peak were quantified using featureCounts (Liao et al., 2014).
Assembly: hg38
Supplementary files format and content: bed, tab-delmited text
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| Submission date |
Aug 15, 2024 |
| Last update date |
Jan 28, 2025 |
| Contact name |
Mark Maienschein-Cline |
| E-mail(s) |
mmaiensc@uic.edu
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| Organization name |
University of Illinois at Chicago
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| Department |
Research Resources Center
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| Lab |
Center for Research Informatics
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| Street address |
1819 W Polk Ave, Rm 336 M/C 789
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60612 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE274958 |
Effects of MRSA and FTY720 S-phosphonate on H3K9 acetylation in human lung endothelial cells |
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| Relations |
| BioSample |
SAMN43207216 |
| SRA |
SRX25717701 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8462829_CS-DW-05.peaks.bed.gz |
867.0 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
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