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| Status |
Public on Feb 23, 2017 |
| Title |
MCF7_H3K27me3_Rep1_TCLSeq |
| Sample type |
SRA |
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| Source name |
Breast cancer cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
tissue: breast
chip antibody: H3K27me3 (Active Motif #39155)
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| Growth protocol |
MCF7 were grown according to ATCC instructions. To produce neurospheres, mice (Black6 from a mixed C57Bl6 and B6C3 background) were euthanized by CO2, decapitated, and their brains immediately removed. The subventricular zone (SVZ) was micro-dissected and stored in ice-cold PBS for further processing. The tissue was digested using Liberase DH (Roche) and DNase I (250 U ml−1) at 37 °C for 20 min followed by trituration. Digested tissue was washed in ice-cold HBSS without calcium and magnesium, filtered through a 40-μm filter and FACS-sorted as Lineage−CD24− cells into neurosphere growth media that is, Neurobasal-A (Invitrogen) supplemented with Glutamax (Life Technologies), 2% B27-A (Invitrogen), mouse recombinant epidermal growth factor (EGF; 20 ng ml−1) and basic fibroblast growth factor (bFGF; 20 ng ml−1) (Shenandoah Biotechnology). Lineage cells were depleted using mouse CD45, CD31, and Ter119 (Biolegend).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP samples,unfixed cells were clarified after enzymmatically digesting and lysing cells, then histone-DNA complexes were isolated with antibody. For TCL samples, cells were permeablized and treated with restriction enzymes to fragment chromatin, then diluted with lysis buffer. Antibody-adapter complexes were added to chromatin fragment solution, then a ligation mix was added to facilitate proximity ligation of the antibody bound adapter. DNA was puried and ligated DNA was amplified prior to library construction.
Libraries were prepared with Illumina's NEXTERA DNA prep kit (FC-121-1031)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Native chromatin TCL-seq using 2,000 cells
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| Data processing |
Sequence reads were generated by Illumina's NextSeq500 platform.
MCF7 reads were aligned to the hg19 genome using Bowtie 1.1.2 with the following parameters: (-5 = 15; -e = 40; -v = 2; -m = 1). Neurosphere reads were aligned to the mm9 genome using Bowtie 2.2.6.2 using -very-fast-local.
Samtools was used to convert and/or filter alignements to BAM files containing all reads with MAPQ > 1
BAM files were then used with Deeptools bamCoverage to produce normalized (RPKM) BigWig signal tracks using 100 bp bins. Reads were extended to 500 bp.
Genome_build: hg19 and mm9
Supplementary_files_format_and_content: BigWig files contain RPKM nnormalized genome-wide signals generated with bamCoverage verison 2.3.6.0. Scores were calculated using 100 bp bins after extending reads to 500 bp.
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| Submission date |
Feb 12, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Mark A Zarnegar |
| E-mail(s) |
zarnegar@stanford.edu
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| Phone |
6264838223
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| Organization name |
Stanford University
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| Department |
Institute for Stem Cell Biology and Regenerative Medicine
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| Lab |
Michael Clarke
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| Street address |
265 Campus Drive
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE94804 |
Genome-wide maps of chromatin state in neurosphere cells and breast cancer cells |
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| Relations |
| BioSample |
SAMN06323345 |
| SRA |
SRX2553929 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2483415_MCF7_TCL_H3K27me3_Rep1.bigwig |
76.5 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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