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| Status |
Public on Nov 08, 2012 |
| Title |
NWBC4_replicate2 |
| Sample type |
SRA |
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| Source name |
healthy white blood cells from adult blood
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| Organism |
Homo sapiens |
| Characteristics |
tissue: adult blood
cell type: healthy white blood cells
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA isolation: Red blood cells (RBCs) were lysed in the RBC lysis solution containing 155 mM ammonium chloride, 10 mM potassium bicarbonate and 1 mM EDTA. White blood cells (WBCs) were isolated as a pellet by centrifugation. The WBC pellet was then lysed in the solution containing 2% sodium dodecyl sulphate and 25 mM EDTA. Proteins were precipitated by adding 1/3 volume of 10 M ammonium acetate and removed as a pellet by centrifugation. DNA was precipitated from the supernatant with isopropanol, washed with 70% ethanol and dissolved in TE (10 mM TRIS pH 8.0, 1 mM EDTA).
Five micrograms of genomic DNA spiked with 5 methylation standards with defined methylation levels of 0, 25, 50, 75, 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 2000 |
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| Description |
Biological replicate 4 of 4.
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| Data processing |
Sequencing tags were mapped to SmaI sites in the human genome (hg18) and signatures corresponding to methylated (starting with CCGGG…) and unmethylated (starting with GGG…) CpG were enumerated for each SmaI/XmaI site.
At each SmaI/XmaI site, let sm be the number of tags corresponding to methylated CpG (i.e., tags starting with CCGGG) and u be the number of tags corresponding to unmethylated CpG (i.e., tags starting with GGG), the methylation value was calculated as 100%*(c*sm/(c*sm+u)), where c is the correction factor calculated based on observed and expected methylation of spiked-in standards.
The value of c for each sample: AML 2.352, CB1 1.911, CB2 2.164, HEL 2.762, K562 2.225, NWBC1_replicate1 1.152, NWBC1_replicate2 1.204, NWBC2_replicate1 2.028, NWBC2_replicate2 1.205, NWBC3 1.748, NWBC4_replicate1 2.137, NWBC4_replicate2 0.858, NWBC4_replicate3 1.352.
Genome_build: hg18
Supplementary_files_format_and_content: The processed data files are tab-delimited text files that contain the methylation values. The columns of these files are: SmaI_ID (SmaI site ID), Chrom (chromosome of the SmaI site), Pos (position of the SmaI site), MetReads (observed number of tags corresponding to methylated CpG, sm), UnmetReads (observed number of tags corresponding to unmethylated CpG, u), Methylation (corrected methylation value).
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| Submission date |
Jul 31, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Jaroslav Jelinek |
| E-mail(s) |
jjelinek@temple.edu
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| Phone |
215-707-7312
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| URL |
http://www.temple.edu
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| Organization name |
Temple University
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| Department |
Fels Institute
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| Lab |
Issa Lab
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| Street address |
3307 North Broad St
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19140 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE39787 |
Conserved DNA methylation patterns in healthy blood cells and extensive changes in leukemia measured by a new quantitative technique. |
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| Relations |
| SRA |
SRX172507 |
| BioSample |
SAMN01096278 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM979030_NWBC4_replicate2.txt.gz |
2.8 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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