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| Status |
Public on May 11, 2012 |
| Title |
CLL131401_RRBS |
| Sample type |
SRA |
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| Source name |
CLL primary B-cells
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Blood
cell type: CD19+ B-cells
tumorgenecity: Tumor
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| Growth protocol |
Primary B-cells were isolated from CLL using Dynal B-cell Isolation Kit (Invitrogen, Carlsbad, CA, USA); Normal B-cell samples were purchased from ALLCells.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
RRBS was performed according to a previously published protocol (Gu et al. 2010; Meissner et al. 2008) with minor modifications. For each sample, 1 g genomic DNA was first digested overnight using 20 units of MspI (New England Biolabs, NEB). The digested DNA was end-repaired and adenylated in a 20 l reaction consisting of 10U of Klenow fragments (exo-, Enzymatics, MA, USA), and 2 l premixed nucleotide triphosphates (1 mM dGTP, 10 mM dATP, 1 mM methylated dCTP). The reaction was incubated at 30 C for 30 min followed by 37 C for an additional 30 min. The methylated Illumina adapters were ligated with adenylated DNA fragments in a 20 l reaction containing 1 l concentrated T4 ligase (Enzymatics) at room temperature for 15 minutes. The ligation products were gel-selected for fragments with insertion sizes of 40 to 120 bp and 120 to 220 base pairs as previously suggested (Gu et al. 2010; Meissner et al. 2008). Bisulfite treatment of the sequencing library was conducted using the EZ DNA methylation kit (Zymo Research, Irvine, CA) according to manufacturer's protocol. The final libraries were generated by PCR amplification of 5 ?m of bisulfite-converted template using PfuTurbo Cx Hotstart polymerase and Illumina pair-end PCR primers (PE1.0 and PE2.0).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
DNA methylation analysis using bisulfite sequencing
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| Data processing |
Alignment: The raw bisulfite sequencing reads were first converted to “unmethylated reads” via the conversion of all Cs to Ts in silico. The unmethylated, converted sequencing reads were then aligned to the two copies of in silico converted human reference genome (hg18) using SOAP2 (http://soap.genomics.org.cn/). The results of both mappings were combined, and any reads mapped against both copies of converted reference were resolved by identifying their strand based on the TCAG ratio and the mapping positions used accordingly
Methylation calculation: The methylation level [(# of methylated)/(# of methylated + # of unmethylated)] was calculated after measuring the amount of methylation (CG->CG) and unmethylation (CG->TG) in the CpG position. Only CpGs that are covered by at least 10 sequencing reads were included.
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| Submission date |
Oct 07, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Huidong Shi |
| E-mail(s) |
[email protected]
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| Phone |
706-721-6000
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| Organization name |
Augusta University
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| Department |
Georgia Cancer Center
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| Lab |
2125 K
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| Street address |
1120 15th Street, CN2138
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| City |
Augusta |
| State/province |
GA |
| ZIP/Postal code |
30912 |
| Country |
USA |
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| Platform ID |
GPL10999 |
| Series (2) |
| GSE32698 |
Genome-wide DNA methylation analysis reveals novel epigenetic changes in chronic lymphocytic leukemia |
| GSE66167 |
Transcriptome analysis and Genome-wide DNA methylation maps in chronic lymphocytic leukemia cells determined by next-generation sequencing |
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| Relations |
| SRA |
SRX100659 |
| BioSample |
SAMN00738561 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM812437_CLL131401.cg.bed.gz |
14.2 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data included within Sample table |
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