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| Status |
Public on Sep 12, 2013 |
| Title |
H3K4me3-a549-3d-treated |
| Sample type |
SRA |
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| Source name |
a549 3D culture
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| Organism |
Homo sapiens |
| Characteristics |
cell line: A549
treated twice with: 10 ng/mL TNF and 2 ng/mL TGF for 48hr each
chip antibody: H3K4me3
chip antibody vendor: Active Motif
chip antibody cat. #: 39159
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| Treatment protocol |
3D cultures were subject to two treatment rounds of 10 ng/mL TNF and 2 ng/mL TGF treatment and incubation for 48 hours each.
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| Growth protocol |
NSCLC lines A549 were obtained from ATCC and maintained as 2D cultures in DMEM (CellGro), 10% FBS (Invitrogen) and penicillin/streptomycin (Invitrogen). For 3D cultures, cells were grown to approximately 80% confluency, trypsinized, resuspended in DMEM/10% FBS and pipeted onto the underside of a sterile 10 cm tissue-culture plate lid and incubated for 48 hours to facilitate cellular aggregation and spheroid formation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Control and EMT-induced cells were cross-linked in 1% formaldehyde. The cross-linking reaction was quenched using 125 mM glycine, and the samples were collected for ChIP-Seq analysis according to the Myers lab protocol as described (Johnson et al.) Approximately 1.2 x 10^7 cells were used per IP, and the DNA was sheared to approximately 400 bp fragments by sonication with a bioruptor.
After DNA recovery, we used standard Illumina protocols and reagents to prepare the ChIP-seq library (Illumina 11257047 rev A).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
processed data file: H3K4me3-a549-3d-differential.tdf
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| Data processing |
Images generated by the Illumina sequencer were initially processed using the the Illumina pipeline.
read alignment: BWA 0.5.9-r16 using the HG19 reference with all default options, matches with smallest edit distance, ties resolved by picking a random.
Sequences that fully or partially overlapped regions with mappability issues, e.g. repetitive sequences, were discarded. These included regions with high false positive rates of enrichments, as identified by Pickrell et al [PMID: 21690102]
Mapped reads were extended to 200bp in the 3’ direction. The the genome was segmented into 200bp bins, and extended tags were assigned to each genomic bin they overlapped giving the per-window tag count (enrichment profile).
The raw enrichment profiles were normalized between the treated (3D) and control (3D) samples using an implementation of the normalization procedure used in the DESeq algorithm [pmid:20979621]. Next, we calculate a differential profile by subtracting the normalized control signal from the treated signal at each genomic window.
To locate putative enhancer sites using epigenetic data: initial putative loci was derived from the enrichment profiles H3K27ac and H3K4me1. The SICER software was used to call peaks of both marks in the epithelial and mesenchymal states, using corresponding panH3 samples as a control Peak calls with gaps less than or equal to 600bp were merged. The final calls were based on a FDR corrected p-value with a cutoff of 0.01. These peaks were subsequently used to delineate enhancer regions. Putative enhancers, for either of the two histone modifications, were centered (“anchored”) on the window that had the highest enrichment within a SICER peak. Sites were filtered to exclude those that overlapped H3K4me3 SICER peaks. H3K4me1 peaks within 1kb of H3K27ac peaks were collapsed to the H3K27ac-based site. The resulting set of 200bp putative enhancer sites were expanded to include the flanking 1kb, to produce a set of 75,937 potential enhancer sites, each 2200bp in length. The initial set of 75,937 putative enhancers was further filtered to enrich for regions with significant epigenetic changes during EMT, yielding a final set of 30,681 differential enhancers.
Genome_build: HG19
Supplementary_files_format_and_content: files ending with "BED" are the results of aligning the "FASTQ" to the reference genome and are in the UCSC BED file format (the 'enhancer-sites.bed' file resulted from computing over all samples in this dataset), files ending with "TDF" (linked to Series records) are the differential enrichment profiles in the IGV "tiled" format suitable for viewing in IGV.
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| Submission date |
Nov 19, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Marcin Piotr Cieslik |
| E-mail(s) |
marcin.cieslik@gmail.com
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| Phone |
4343262626
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| Fax |
4343262626
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| Organization name |
University of Virginia
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| Street address |
400 Stribling Ave Ext
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| City |
Charlottesville |
| State/province |
VA |
| ZIP/Postal code |
22903 |
| Country |
USA |
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| Platform ID |
GPL10999 |
| Series (2) |
| GSE42374 |
Histone modification analysis in TGFbeta/TNFalpha treated A549 spheroid cultures |
| GSE42375 |
chromatin reprogramming and differential gene expression in a model of EMT (spheroid A549 treated with TGF/TNF) |
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| Relations |
| SRA |
SRX205087 |
| BioSample |
SAMN01816048 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1038456_H3K4me3-a549-3d-treated.bed.gz |
290.4 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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