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| Status |
Public on Jun 13, 2012 |
| Title |
103 year-old male Caucasian |
| Sample type |
SRA |
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| Source name |
a centenarian
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| Organism |
Homo sapiens |
| Characteristics |
racial classification: Caucasian
gender: male
age: 103 years
source tissue: Peripheral blood
cell type: CD4+T Cells
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| Treatment protocol |
Peripheral blood was obtained from a healthy donor (age 103) and from umbilical cord blood from a newborn. Peripheral blood mononuclear cells (PBMC) were extracted using a Ficoll gradient. To separate CD19 and CD4 positive cells CD19 MicroBeads and CD4+ T cell Isolation kit II (Miltenyi Biotec) was applied following the manufacturerâs instructions. DNA was extracted using Phenol:Chloroform:Isoamylalcohol (Sigma), respectively.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For normal BS-seq library constructing, 10ug genomic DNA was fragmented using a Covaris sonication system (Covaris S2). Following fragmentation, libraries were constructed using the Illumina Paired-End protocol consisting of end repair, <A> base addition and methylated-adaptor ligation. Ligated DNA was bisulfite converted using the EZ DNA Methylation-Gold kit (ZYMO). Different insert size were excised from the same lane of a 2% TAE agarose gel. Normally, three bands are excised corresponding to DNA insert sizes of 80-100 bp, 100-120 bp to 120-150bp. Products were purified by using QIAquick Gel Extraction kit (Qiagen) and amplified by PCR. PCR was carried out in a final reaction volume of 50ul consisting of 20ul purified DNA, 4ul 2.5 mM dNTP, 5 ul 10X buffer, 0.5 ul JumpStart Taq DNA polymerase, 2ul 10uM PCR primers and 37.5 ul ÎltraPure TM Water and the following thermal cycling program: 94C 30 s, 10 cycles of 94C 30 s, 60C 30 s, 72C 30 s then prolong with 1 min at 72C. PCR products were sequenced with an Illumina genome analyzer. The reads generated by Illumina sequencing were aligned to the reference genome using SOAPaligner2. The alignment and methylation estimation was performed as described before (Li, Y. et al. PLoS Biol 8, e1000533 (2010)).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
Y103
Whole Genome Bisulphite sequencing : EZ DNA Methylation Gold kit (ZIMO)
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| Data processing |
Read mapping: Two reference sequences were prepared based on the hg19 human reference; referenceC2T had the C residues replaced by T's, and referenceG2A had the G's replaced by A's. Two sets of reads were generated in the same way; setC2T with the C's replaced by T's, and setG2A with the G's replaced by A's. Both sets of reads were aligned to each of the two references, producing four sets of mappings. This mapping strategy allowed reads to be aligned irrespective of the methylation status of any C residues. The alignment was performed using the GEM alignment software allowing up to 4 mismatches in high quality bases (bases with quality scores > 25). Uniquely mapping read pairs were used to generate an empirical distribution of insert sizes of each of the libraries, and this is information was used to select read pairs with unique compatible mappings.
The bisulfite conversion followed by a PCR step generates 4 different strands; the original + and - strands along with their complements. Reads derived from the original + strand (and its complement) will align to referenceC2T, while reads from the original - strand (and complement) will align to referenceG2A. Both members of a read pair are derived from the same original strand, and >99% of mapped reads could have the original strand assigned unambiguously. By keeping the information on the original strand origin of each read pair, the methylation status of C residues on either of the two original strands can be inferred along with the underlying genotypes, because any conversion due to the bisulphite treatment will only be seen on read pairs deriving from one of the two original strands.
Methylation call files were generated using Bismark methylation call procedure from the bam aligned files and then processed as a bed files with columns showing : Chromosome, initial and end position of this Cpg and its methylation value
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| Submission date |
Aug 08, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Manel Esteller |
| Organization name |
IDIBELL
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| Department |
PEBC
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| Lab |
Cancer Epigenetics
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| Street address |
Hospital Duran i Reynals Av. Gran Via s/n km, 2.7
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| City |
L'Hospitalet de Llobregat |
| State/province |
Barcelona |
| ZIP/Postal code |
08908 |
| Country |
Spain |
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| Platform ID |
GPL9052 |
| Series (1) |
| GSE31263 |
The DNA methylomes of a newborn and a centenarian |
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| Relations |
| SRA |
SRX091573 |
| BioSample |
SAMN00709113 |
| Named Annotation |
GSM774849_103_CpG.bed.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM774849_103_CpG.bed.gz |
70.7 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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