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| Status |
Public on Feb 21, 2015 |
| Title |
SC4_RRBS |
| Sample type |
SRA |
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| Source name |
Normal CD19+ B-cells
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| Organism |
Homo sapiens |
| Characteristics |
tissues: Blood
cell type: CD19+ B-cells
tissue: Normal
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| Treatment protocol |
No treatment invovled.
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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 |
Genomic DNA was extracted using Qiagen kit
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 HiSeq 2000 |
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| Description |
DNA methylation analysis using bisulfite sequencing
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| Data processing |
To map the sequencing reads from RRBS, we extracted sequenceable regions from the human genome (hg18) that were within 100bp from the MspI sites at both ends. Each sequenceable region was indexed by converting all C’s and G’s to T’s and A’s, respectively, i.e., two different reference databases. Bowtie was used to map the cleaned reads to each of the two reference databases after converting all C’s to T’s.
For each read, an in-house script computed the best of all alignments for the different loci using two different reference databases based on the number of mismatches after realigning the original read and reference sequences. The script also determined the methylation status of each cytosine residue by comparing the bisulfite-converted sequence to the reference sequence.
Another in-house script piled reads for each cytosine in the reference genome and counted the numbers of reads that contained methylated and unmethylated cytosines, respectively. Finally the methylation of each CpG site was defined as the fraction of methylated reads to that of methylated and unmethylated reads combined. CpGs with < 5 reads were filtered out for further analyses.
Genome_build: hg18
Supplementary_files_format_and_content: BED format, Methylation level at each CpG sites ( a value between 0 and 1)
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| Submission date |
Feb 19, 2015 |
| 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 |
GPL11154 |
| Series (2) |
| GSE66121 |
Genome-wide DNA methylation maps in chronic lymphocytic leukemia cells determined by next-generation sequencing (RRBS) |
| GSE66167 |
Transcriptome analysis and Genome-wide DNA methylation maps in chronic lymphocytic leukemia cells determined by next-generation sequencing |
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| Relations |
| BioSample |
SAMN03360408 |
| SRA |
SRX885212 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1614762_SC4.cg.bed.gz |
32.4 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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