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| Status |
Public on Aug 12, 2014 |
| Title |
MCF7 Input CHART-seq Ethanol treatment, rep2 |
| Sample type |
SRA |
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| Source name |
MCF-7, ethanol vehicle, CHART input
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7
cell type: mammary adenocarcinoma cell line
treatment: ethanol vehicle
capture oligo: none
antibody: N/A
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For CHART-seq, nuclei were isolated from 10^8 MCF-7 cells (ATCC HTB-22) that were crosslinked with 1% formaldehyde for 10 minutes. Nuclei were further crosslinked with 3% formaldehyde for 30 minutes. Chromatin was sheared to a fragment size between 2 kb and 10 kb. Sheared chromatin was incubated overnight with biotinylated DNA oligonucleotides with complementarity to either the NEAT1 or MALAT1 RNAs, and subsequently captured with streptavidin beads. After incubation and washing, captured materials were eluted using RNaseH and subjected to crosslink reversal. DNA was purified by ethanol precipitation and subjected to library construction.
Genomic DNA isolated by CHART enrichment, as well as input DNA, was fragmented using sonication. Sonicated DNA was then subjected to end-repair, A-tailing, ligation to universal adapters, amplification with indexed primers, and size selection using SPRI beads.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
CHART Input DNA.
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| Data processing |
Library strategy: CHART-Seq
Base-calling was performed using Illumina Real Time Analysis software.
Reads were aligned using Bowtie v 0.12.5 with the following options: -m 1 -v 2 -k 1 -X 2000 --best --strata.
After alignment, reads were processed and filtered according to the SPP guidelines, where the genomic positions with a number of mapped tags above the significance threshold of z-score = 7 were identified as anomalous, and the tags mapped to such positions were discarded.
The coverage for each sample represents frequencies of the paired-end reads spanning over a given genomic position, which were corrected for library size and subsequently input normalized.
For analysis of CHART-seq and PSF paired-end ChIP-seq data from untreated MCF-7 cells, coverage was normalized by input as n_norm = [(n+1)/(ni+1)] * [Ni/N], where n, ni, N, and Ni are positional coverage in experiment and input, and total genome coverage in experiment and input, respectively. The resulting normalized coverage files are included in this dataset in bigwig format.
Input-subtracted, normalized read densities of flavopiridol, E2, and respective vehicle CHART-seq were generated using SPP with a bandwidth of 1kb and a step size of 500 bp and are included in this dataset in bigwig format.
Genome_build: GRCh37 (hg19)
Supplementary_files_format_and_content: All processed data files are in bigwig format and represent input-normalized CHART-seq or ChIP-seq coverage.
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| Submission date |
Jun 12, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Ruslan Sadreyev |
| Organization name |
Massachusetts General Hospital
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| Department |
Molecular Biology
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| Lab |
Bioinfomatics
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| Street address |
185 Cambridge Street
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| City |
Boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02114 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE58444 |
The long noncoding RNAs NEAT1 and MALAT1 bind active chromatin sites |
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| Relations |
| BioSample |
SAMN02853024 |
| SRA |
SRX593765 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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