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| Status |
Public on Mar 16, 2017 |
| Title |
MCF7 H3K4me2 S phase |
| Sample type |
SRA |
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| Source name |
MCF7 breast cancer cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
cell type: breast cancer
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| Treatment protocol |
Cells are synchronized to three different cell cycle stages: G0/G1, S and M phase with hormone starvation, double thymidine and thymidine/Noco treatment respectively.
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| Growth protocol |
The MCF-7 cells were obtained from ATCC and cultured in DMEM media supplemented with 10% FBS, 1% Pen-Strep and 1% Glutamine in a 5% CO2 humidified incubator.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
GRO-seq: Nuclear run-on RNA was further purified with TURBO DNA-freeTM kit (Life Technologies, AM1907) to remove residue DNA contamination. RNA-seq: Total RNA was extracted from synchronized MCF-7 cells with TRIZOL REAGENT (Life Technologies, 15596026) and fragmentized with sonication. ChIP-seq: Chromatin from synchronized MCF-7 cells was digested with Nuclease micrococcal from Staphylococcus aureus (Sigma Aldrich, N3755-50UN) and dialyzed with Slide-A-Lyzer Dialysis Cassettes (Thermo Fisher Scientific, PI66380). Dynabeads Protein A (Life Technologies, 10002D) and G (Life Technologies, 10004D) were used for immunoprecipitation with antibodies against H3K4me2 (EMD Millipore, 07-030) and H3K27ac (Abcam, ab4729).
GRO-seq and RNA-seq libraries were constructed with Encore Complete RNA-Seq DR Multiplex System 1-8 (Nugen Technologies lnc, 0333-32). ChIP-seq libraries were constructed with ThruPLEX DNA-seq kit (Rubicon) and sequenced to 50bp with Illumina HiSeq machine.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Two replicates are merged:
H3K4me2 S phase rep1
H3K4me2 S phase rep2
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| Data processing |
GRO-seq reads were mapped to the human genome (hg19) using Bowtie with default parameters.
RNA-seq reads were aligned to human genome (hg19) with tophat (v2.1.1). ChIP-seq and Dnase-seq reads were aligned to human genome (hg19) with Bowtie.
MACS2 was used for ChIP-seq and Dnase-seq peak calling with the parameter "–SPMR" on to produce bedGraph files, which were then converted to bigwiggle files
Htseq (v0.5.4p3) was used to get gene level read counts for GRO-seq data and DESeq2 (v1.8.2) was used for differential gene analysis; GFOLD (v1.1.3) was used to generate gene level read count and carry out differential expression analysis.
bedGraph files for GRO-seq were generated using genomeCoverageBed function
in the BEDTools(v2.20.1) suite with total signal of 10 million, replicates were then combined before converted to bigwiggle files.
Genome_build: hg19
Supplementary_files_format_and_content: bigwiggle files were converted from bedGraph with bedGraphToBigWig function in BLAT suite. For GRO-seq and RNA-seq data, score equals to reads per 20 million; for ChIP-seq and Dnase-seq data, score equals to reads per million.
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| Submission date |
Feb 03, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Housheng Hansen He |
| E-mail(s) |
Hansen.He@uhnresearch.ca
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| Organization name |
University of Toronto
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| Lab |
He lab
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| Street address |
101 College Street
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 1L7 |
| Country |
Canada |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE94479 |
Transcriptional landscape of the human cell cycle |
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| Relations |
| BioSample |
SAMN06294277 |
| SRA |
SRX2537033 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2476259_S_H3K4me2_merged_treat_pileup.bw |
806.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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