|
| Status |
Public on Dec 29, 2009 |
| Title |
Newborn Human Foreskin Fibroblasts: Bisulfite sequencing |
| Sample type |
SRA |
| |
|
| Source name |
Primary fibroblast
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Newborn Human Foreskin Fibroblasts
|
| Growth protocol |
WA09 hESC line was cultured feeder-free on MatrigelTM (Becton-Dickinson) in StemPro TM medium (Lifetech, Inc.), and passaged with Accutase (Lifetech, Inc.). They were harvested at passage 41. hESC-derived fibroblasts (hESC-Fibro) were derived as a stable proliferating population from spontaneously differentiating passage 40 WA09 hESC cells (Gonzalez et al., 2008); after their differentiation they were expanded in two batches, to passage 11 and passage 13 before harvesting. The neonatal fibroblast line (Fibro) cell line was obtained from GlobalStem, Inc (catalog number GSC-3002, Newborn Human Foreskin Fibroblasts, untreated) and was harvested for analysis at passage 13. All cell lines were cultured at 37°C, ambient oxygen, and 5% CO2. The hESC-Fibro and Fibro cell lines were cultured in DMEM+10%FBS and passaged with trypsin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA were extracted from the individual cell lines and fragmented via nebulization to sizes between 300 bp to 500 bp. The fragmented DNA was end polished and ligated with Illumina methylated PE adaptors followed by two consecutive bisulfite treatments using the EpiTect Bisulfite Kit (Qiagen) to ensure maximal conversion rate. The bisulfite treated DNA was enriched by 10 cycles of PCR with primers complementary to the adaptor sequences by uracil-insensitive Taq polymerase (Pfu Turbo Cx Hotstart DNA Polymerase; Stratagene). Sizes between 250 bp to 300 bp (including the adaptors) were selected and sequenced with the Illumina Genome Analyzer using the PE sequencing method at 75bp read length
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Fibro DNA methylation mapping data
|
| Data processing |
Alignment: Sequence reads were first processed by Illumina Genome Analyzer pipeline for base calling and quality filtering. Reads that passed filtering were aligned to human reference genome, Hg18, via SOAP2 alignment program with less than 2 mismatches. To accommodate the conversion of unmethylated cytosines to thymines by bisulfite conversion, we used a "reduced", three-letter alphabet comprising A, G and {C or T} to represent the genome.
Methylation levels of a C within an aligned read were gauged by the ratio of reads that contained a methylated C at that location vs. all the reads that covered the location.
|
| |
|
| Submission date |
Dec 28, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Chia-Lin Wei |
| E-mail(s) |
weicl@gis.a-star.edu.sg
|
| Phone |
65 64788074
|
| Fax |
65 64789059
|
| URL |
http://www.gis.a-star.edu.sg/internet/site/investigators.php?f=cv&user_id=9
|
| Organization name |
Genome Institute of Singapore
|
| Department |
Genome Technology & Biology
|
| Street address |
60 Biopolis Street #02-01
|
| City |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL9115 |
| Series (1) |
| GSE19418 |
Dynamic Changes in the Human Methylome During Differentiation |
|
| Relations |
| Reanalyzed by |
GSE46644 |
| SRA |
SRX015769 |
| SRA |
SRX015770 |
| SRA |
SRX015771 |
| SRA |
SRX015772 |
| BioSample |
SAMN00007499 |
