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| Status |
Public on Feb 08, 2017 |
| Title |
NBE_1013140_DREAM |
| Sample type |
SRA |
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| Source name |
normal breast epithelial cells
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| Organism |
Homo sapiens |
| Characteristics |
tissue of origin: breast
disease: adjacent normal tissue from 69 year old patient with lobular carcinoma in situ
gender: female
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| Treatment protocol |
none
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| Growth protocol |
MCF7 cell line was cultured in DMEM medium with 10% fetal bovine serum, normal breast epithelial cells were grown in 1:1 DMEM/F12 medium with 2.4 g/L sodium bicarbonate, 5% chelated horse serum, 20 ng/ml EGF (BD Biosciences), 100 ng/ml cholera toxin (Sigma-Aldrich), 10 mg/L insulin (Sigma-Aldrich), 0.5 mg/L hydrocortisone (Sigma-Aldrich), and 0.04 mM calcium chloride (Sigma-Aldrich)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
phenol-chloroform
Five micrograms of genomic DNA spiked with 500 ng of methylation standards with defined methylation levels of 0, 25, 50, and 100% were digested with 100 units of SmaI endonuclease (NEB) for 3 hours at 25°C. Subsequently, 100 units of XmaI endonuclease (NEB) were added and the digestion was continued for an additional 16 hours at 37°C. Digested DNA was purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA) and eluted in TRIS-HCl 10 mM pH 8.5 (EB). Eluted DNA was supplemented with NEB buffer #2, dCTP, dGTP and dATP (0.4 mM final concentration of each), 15 units of Klenow Fragment (3'→5' exonuclease deficient) DNA polymerase (NEB) and incubated for 30 minutes at 37°C. This step filled in the recesses at 3' DNA ends created by XmaI digestion and added 3' dA tails to all fragments. Illumina paired end (Quail et al. 2008) or barcoded Truseq (Illumina, San Diego, CA) sequencing adapters were then ligated at 10:1 adapter:fragment ratio using Rapid T4 DNA ligase (Enzymatics, Beverly, MA). The ligation mix was size selected by electrophoresis in 2% agarose. Two slices corresponding to 250-350 bp and 350-500 bp sizes based on a 100 bp DNA ladder (NEB) were cut out and DNA was extracted from agarose. DNA eluted from the slices was separately amplified with Illumina paired end PCR primers (Quail et al. 2008) using iProof high-fidelity DNA polymerase (Bio-Rad Laboratories, Hercules, CA) and 18 cycles of amplification. Resulting sequencing libraries were purified with AMPure magnetic beads (Agencourt, Beverly, MA).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Adjacent normal breast tissue was isolated from patients with breast cancer, then epithelial cells were isolated and grown in culture for a few passages before DNA isolation
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| Data processing |
Library strategy: DREAM
Illumina HiSeq2500 paired end sequencing
Raw sequencing data were converted to fastq.gz files.
bowtie2 --sensitive alignment of the reads in fastq files to hg19 and generating SAM files
generating sorted bam files from the sam files with samtools
counting methylated CCGGG signatures and unmethylated GGG at the reads aligned to CCCGGG sites
Adjusting methylation values based on the spiked in standards. At each SmaI/XmaI site, let Xma be the number of reads corresponding to methylated CpG (i.e., reads starting with CCGGG) and Sma be the number of reads corresponding to unmethylated CpG (i.e., reads starting with GGG), the methylation value was calculated as 100%*(c*Xma/(c*Xma+Sma)), where c=2.342 was the correction factor calculated based on observed and expected methylation of spiked-in standards.
Genome_build: hg19
Supplementary_files_format_and_content: The processed data are in tab-delimited text file. The columns of the file are: ID_SmaI_hg19 (CCCGGG site ID), chromosome (chromosome of the CCCGGG site), position (position of the CCCGGG site), MethReads (observed number of reads corresponding to methylated CpG, Xma), UnmethReads (observed number of reads corresponding to unmethylated CpG, Sma), RawMethylation (unadjusted methylation value), AdjMethylation (methylation value adjusted based on the spiked in standards).
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| Submission date |
Nov 11, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Takahiro Sato |
| Organization name |
Temple University
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| Department |
Fels Institute for Cancer Research and Molecular Biology
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| Street address |
3307 North Broad Street
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19140 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE74910 |
Digital Restriction Enzyme Analysis of Methylation (DREAM) of normal breast epithelial cells and MCF7 genomic DNA |
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| Relations |
| BioSample |
SAMN04260133 |
| SRA |
SRX1426955 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1937244_NBE_1013140.txt.gz |
3.8 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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