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| Status |
Public on Jul 03, 2014 |
| Title |
MCF7_BisulfiteSeq |
| Sample type |
SRA |
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| Source name |
MCF-7 WT
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| Organism |
Homo sapiens |
| Characteristics |
genetic manipulation: WT
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Pelleted cells were resuspended in digestion buffer (100mM NaCl, 10mM Tris-HCl pH8, 15mM EDTA pH8, 0.5% SDS, 0.1mg/ml Proteinase K) and incubated at 55°C for 12-18 hr. DNA was isolated by two times extraction with 5 ml phenol/chloroform/isoamyl alcohol, and the aqueous top layer was transferred to a new tube. To precipitate DNA, 1/2 volume 7.5M ammonium acetate and 2 volumes 100% ethanol were added. After spinning, the supernatant was discarded, the DNA pellet washed with 70% ethanol, dried, and resuspended in water.
Library construction was performed according to Kulis et al. (2012; Nature Genetics). In short: We spiked genomic DNA (1 or 2 μg) with unmethylated λ DNA (5 ng of λ DNA per μg of genomic DNA) (Promega). We sheared DNA by sonication to 50–500 bp with a Covaris E220 and selected 150- to 300-bp fragments using AMPure XP beads (Agencourt Bioscience Corp.). We constructed genomic DNA libraries using the TruSeq Sample Preparation kit (Illumina Inc.) following the Illumina standard protocol: the ends of the fragments were repaired, an adenine was added at the 3′ extremities of the fragments and Illumina TruSeq adapters were ligated at each extremity. After adaptor ligation, we treated DNA with sodium bisulfite using the EpiTect Bisulfite kit (Qiagen) following the manufacturer’s instructions for formalin-fixed and paraffin-embedded (FFPE) tissue samples. We performed two rounds of conversion to get >99% conversion. We enriched adaptor-ligated DNA through seven cycles of PCR using the PfuTurboCx Hotstart DNA polymerase (Stratagene). We monitored library quality using the Agilent 2100 BioAnalyzer (Agilent) and determined the concentration of viable sequencing fragments (molecules carrying adapters at both extremities) by quantitative PCR using the Library Quantification Kit from KAPA Biosystem. We performed paired-end DNA sequencing (two reads of 100 bp each) using the Illumina Hi-Seq 2000.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Cluster generation and sequencing were performed using the Illumina GAIIx or HiSeq2000 genome analyzer. The 36 (GAIIx) or 43 bp tags (HiSeq2000) were mapped to the reference human genome hg19 (NCBI build 37), using the BWA allowing one mismatch. The image files were processed to extract DNA sequence data. Only uniquely mapped-reads were used for data analysis and visualization. The output data were converted to bam files for downstream analysis and bigWig (bw) files for viewing the data in the UCSC Genome Browser.
FASTQ sequence files from bisulfite-seq were aligned using RMAPBS (Smith et al., 2009; Smith et al., in prep) allowing a maximum of 10 mismatches for efficient mapping of reads containing bisulfite converted unmethylated cytosines. Cytosine methylation levels were determined using the standard new pipeline within RMAPBS (Methpipe; Andrew Smith, in prep). In short, both mates of the paired-end sequencing were mapped separately by RMAPBS. If mated reads mapped within 300 bp, the reads were merged and included for analysis. Mated reads mapping with a distance of more than 300 bp were excluded from further analysis. Mated reads mapped within 300 bp, but mapping equally well on multiple positions on the genome, were discarded. If multiple mated reads mapped on exactly the same genomic coordinates, all but one (at random) were discarded. Symmetric cytosines (within a CpG context) on both forward and reverse strands were combined. Cytosine methylation level was called per individual C as: #C/(#C+#T).
Genome_build: hg19
Supplementary_files_format_and_content: BigWig were generated by counting the number of sequence reads (directionally extended to a final 300 bp) per 10-bp genomic window. Methylation density represents methylation level per 50-bp genomic windows
Supplementary_files_format_and_content: Cytosine methylation levels within CpG context are present in the MCF7_CpG_methcounts.bed.gz
Supplementary_files_format_and_content: Peak-calling was performed with MACS 2.0 tool against a reference input sample from the cell line expressing TTE-MBD2
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| Submission date |
Feb 13, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
H.G. Stunnenberg |
| E-mail(s) |
h.stunnenberg@ncmls.ru.nl
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| Phone |
+31-24-3610520
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| Organization name |
Radboud University
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| Department |
Department of Molecular Biology (274)
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| Street address |
Geert Grooteplein 28
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| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE54693 |
Genome wide profiling of MBD2 binding |
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| Relations |
| BioSample |
SAMN02642023 |
| SRA |
SRX471219 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1328112_MCF7_CpG_methcounts.bed.gz |
262.1 Mb |
(ftp)(http) |
BED |
| GSM1328112_methdens_hg19_50bp_win.bw |
934.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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