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| Status |
Public on Jul 08, 2009 |
| Title |
CHPSKN1_Biulfite_seq |
| Sample type |
SRA |
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| Source name |
CHP-SKN1 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Human primary dermal fibroblasts
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| Growth protocol |
CHP-SKN1 cells were grown in 15 cm plates with DMEM medium containing 20% FBS supplemented with L-glutamine, non-essential amino acids and penicillin/streptomycin. MDA-MB-231 cells were grown in DMEM containing 15% FBS, L-glutamine, non-essential amino acids, and penicillin/streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Purified cell-line DNA was randomly fragmented by sonication and subsequently treated with a mixture of T4 DNA Polymerase, E. coli DNA polymerase I Klenow fragment, and T4 polynucleotide kinase to repair, blunt and phosphorylate ends according to the manufacturer’s instructions (Illumina). The repaired DNA fragments were subsequently 3’ adenylated using Klenow exo- fragment (Illumina). After each step, the DNA was recovered using the QIAquick PCR Purification kit (Qiagen). Adenylated fragments were ligated to Illumina-compatible paired-end adaptors synthesized with 5’-methyl-cytosine instead of cytosine (Illumina) and fragments ranging from 150-300 bp were extracted by gel purification using the QIAquick gel extraction kit (Qiagen) followed by elution in 30ul elution buffer. Following size selection and gel purification, the adapter-ligated DNA was divided into two separate reactions to ensure optimal DNA concentration for subsequent cytosine conversion reactions. Fragments were denatured and treated with sodium bisulfite using the EZ DNA methylation gold kit according to the manufacturer’s instructions (Zymo). Lastly, the sample was desulfonated and the converted, adaptor-ligated fragments were PCR enriched using paired-end adaptor-compatible primers 1.0 and 2.0 (Illumina) and Expand High Fidelity PLUS PCR system (Roche), a specialized polymerase capable of amplifying the highly denatured, uracil-rich templates. PCR reactions contained 1x Expand HiFi PLUS reaction buffer, 200μM each dNTP, 1μM each primer, 2.5mM MgCl2, and 2.5 U Expand HiFi PLUS enzyme. The reactions were performed under the following conditions: 94ºC for 2 minutes, 25 cycles of 94ºC for 15 seconds, 65ºC for 30 seconds and 72ºC for 30 seconds, followed by 72ºC for 7 minutes. Following amplification, the samples were hybridized to capture arrays and captured fragments were recovered and sequenced.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Targeted bisulfite sequencing
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| Data processing |
Alignment: Sequence reads from BS capture experiments were mapped to the human genome (March 2006) with the RMAPBS program, freely available from the authors as Open Source software under the GNU Public License. Reads mapped ambiguously to multiple locations in the genome were ignored in subsequent analysis.
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| Submission date |
Jul 08, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Emily Hodges |
| E-mail(s) |
hodges@cshl.edu
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| Organization name |
Cold Spring Harbor Laboratory
|
| Street address |
1 Bungtown Road
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| City |
Cold Spring Harbor |
| State/province |
NY |
| ZIP/Postal code |
11724 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (1) |
| GSE17001 |
High definition profiling of mammalian DNA methylation by array capture and single molecule bisulfite sequencing |
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| Relations |
| SRA |
SRX007139 |
| BioSample |
SAMN02196673 |
| Named Annotation |
GSM425461_CHPSKN1_BS_aligned.bed.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM425461_CHPSKN1_BS_aligned.bed.gz |
216.2 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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