 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jan 02, 2014 |
| Title |
Genome-wide CpG methylation during osteogenic and myogenic differentiation from adipose- derived stem cells. |
| Organism |
Homo sapiens |
| Experiment type |
Methylation profiling by array
|
| Summary |
In the current study, we have performed a high-throughput CpG methylation analysis of well characterized and defined populations of human adipose-derived stem cells (hASCs) before and after in vitro induction of osteogenic and myogenic differentiation that allows identifying DNA methylation- regulated differentiation genes. We have also address the extent of the epigenetic programming of hASCs- derived differentiated cells by comparing the methylation profiling of these cells with their somatic counterparts from primary tissues. Finally, we also compared the patterns of CpG methylation of hASCs (and their derivatives) with the methylation profiles of myosarcoma and osteosrcoma cell lines. All the CpG methylation studies have been performed with the Infinium 27K methylation arrays (from Illumina).
|
| |
|
| Overall design |
DNA from adipose –derived stem cells (n=4), in vitro induced myocytes (n=3), in vitro induced osteocytes (n=3), primary osteocytes obtained from ribs (n=1), primary myocytes (n=1), osteosarcoma cell line (MG63) and myosarcoma cell lines (Te 32.T and RD) was isolated applying the QIAamp® DNA Mini Kit (Qiagen Iberia, Spain). Microarray- based DNA methylation profiling was performed with the HumanMethylation27 BeadChip Infinium Methylation Arrays® (Illumina, Inc.).The panel was designed to compare the DNA methylation status of each group of samples, which allow interrogating 27,578 CpG loci covering 14,495 genes at single-nucleotide resolution by typing bisulfite-converted DNA. The sequences included in the panel are derived from the well-annotated NCBI CCDS database (Genome Build 36) and is supplemented with more than 1,000 cancer-related genes described in published literature. Probe content has been enriched to deeply cover more than 150 well-established cancer genes known to show differential methylation patterns. Methylation array content also targets the promoter regions of 110 miRNA genes. Methylation arrays were performed as follows. Briefly, bisulfite conversion of 1 μg of genomic DNA was done using the CpGenomicTM DNA Modification Kit (Intergen Company, Purchase, NY, USA). After sodium bisulfite treatment, the remaining assay steps used Infinium technology and using Illumina-supplied reagents and conditions. A thermocycling program with a short denaturation step included for bisulfite conversion (16 cycles of 95ºC for 30 seconds followed by 50ºC for 1 hour) was performed to improve bisulfite conversion efficiency. After bisulfite conversion, each sample was whole-genome amplified (WGA) and enzymatically fragmented. The bisulfite-converted WGA-DNA samples were purified and applied to the BeadChips. During hybridization, the WGA-DNA molecules anneal to locus-specific DNA oligomers linked to individual bead types. The two bead types correspond to each CpG locus -one to the methylated and the other to the unmethylated state. Allele-specific primer annealing is followed by single-base extension using DNP- and Biotin-labeled dNTPs. Both bead types for the same CpG locus will incorporate the same type of labelled nucleotide, determined by the base preceding the interrogated cytosine in the CpG locus, and therefore will be detected in the same colour channel. After extension, the array is fluorescently stained, scanned, and the intensities of the unmethylated and methylated bead types measured. DNA methylation values, described as beta values, are recorded for each locus in each sample via BeadStudio software. DNA methylation beta values are continuous variables between 0 (completely unmethylated) and 1 (completely methylated), representing the ratio of the intensity of the methylated bead type to the combined locus intensity.
|
| |
|
| Contributor(s) |
Esteller M, Berdasco M |
| Citation(s) |
23031258, 34440035 |
| Submission date |
Nov 22, 2011 |
| Last update date |
Aug 31, 2021 |
| Contact name |
Antonio Gómez |
| E-mail(s) |
agomezm@idibell.cat
|
| URL |
http://www.pebc.cat/
|
| Organization name |
IDIBELL
|
| Department |
Epigenetics and Biology of Cancer Program
|
| Lab |
PEBC
|
| Street address |
Av. Gran Vía s/n km. 2,7.
|
| City |
L'Hospitalet de Llobregat |
| State/province |
Barcelona |
| ZIP/Postal code |
08907 |
| Country |
Spain |
| |
|
| Platforms (1) |
| GPL8490 |
Illumina HumanMethylation27 BeadChip (HumanMethylation27_270596_v.1.2) |
|
| Samples (16)
|
| GSM838506 |
genomic DNA from adipose- derived stem cells (donor 1) |
| GSM838507 |
genomic DNA from adipose- derived stem cells (donor 2) |
| GSM838508 |
genomic DNA from adipose- derived stem cells (donor 3) |
| GSM838509 |
genomic DNA from adipose- derived stem cells (donor 5) |
| GSM838510 |
genomic DNA from in vitro induced osteocytes (donor 1) |
| GSM838511 |
genomic DNA from in vitro induced osteocytes (donor 2) |
| GSM838512 |
genomic DNA from in vitro induced osteocytes (donor 3) |
| GSM838513 |
genomic DNA from in vitro induced myocytes (donor 1) |
| GSM838514 |
genomic DNA from in vitro induced myocytes (donor 2) |
| GSM838515 |
genomic DNA from in vitro induced myocytes (donor 3) |
| GSM838516 |
genomic DNA from osteosarcoma cell line (ATCC CRL-1427) |
| GSM838517 |
genomic DNA from rhabdomyosarcoma (ATCC CRL-7731) |
| GSM838518 |
genomic DNA from rhabdomyosarcoma (ATCC CCL-136) |
| GSM838519 |
genomic DNA from primary osteocytes (donor 1) |
| GSM838520 |
genomic DNA from primary myocytes (donor 1) |
| GSM838521 |
genomic DNA from primary myocytes (donor 2) |
|
| Relations |
| BioProject |
PRJNA148337 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE33896_RAW.tar |
5.8 Mb |
(http)(custom) |
TAR |
| GSE33896_signals.txt.gz |
2.4 Mb |
(ftp)(http) |
TXT |
| Processed data included within Sample table |
|
|
|
|
 |
Less...
More...