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| Status |
Public on Jan 01, 2015 |
| Title |
SclKO_MES_RRBS |
| Sample type |
SRA |
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| Source name |
ES cell derived day4 EB (embryoid body) Flk1+ mesodermal cells
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| Organism |
Mus musculus |
| Characteristics |
cell-line: SclKO J1 mESC
cell type: SclKO ES cell derived day4 EB (embryoid body) Flk1+ mesodermal cells
cell-line: SclKO J1 mESC
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| Treatment protocol |
EB formation, enrichment for Flk1+ cells at day 4
ES cells were differentiated into Flk1+ cells by 2 days of culture in pre-EB media (IMDM with the addition of L-glutamine, 1% of penicillin and streptomycine, 1.5 x 10-4 M monothiolglycerol (MTG), 15% of FBS and 10ng/ml LIF; followed by plateing 100 000 cells per well to low attachment 6-well plates (Costar) in EB media ((IMDM with the addition of 15% of FBS, L-glutamine, 1% of penicillin and streptomycine, 1.5 x 10-4 M MTG, 3mg/ml human transferrin (Roche) and 2.5mg/ml ascorbic acid (Sigma) and clulturing the cells for 4 days.
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| Growth protocol |
Standard ES cell culture media with DMEM (Cellgro), 15% serum (Hyclone or Omega) and 10ng/ml LIF (Millipore) and gelatin coated dishes were used to maintain WT and SclKO (Porcher et al., 1996) ES cells. MEL cells were grown in RPMI 1640 with L-Glutamine plus10% of FBS,1% of penicillin and streptomycine.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using Allprep DNA/RNA Mini Kit (Quiagen) and quanitated with QUBIT (Life technology). RRBS libraries were generated starting from 240-450 ng of genomic DNA as previously described (Meissner et al., 2005) with minor modifications. Briefly, MspI- generated fragments were end-repaired and adenylated before ligation with Illumina TruSeq adapters. DNA purifications of each enzymatic reaction as well as size-selection of adapter-ligated fragments ranging from 200-350bp were carried out using AMPure XP beads (Beckman Coulter). Bisulfite conversion was performed twice with EpiTect kit (QIAGEN) in order to optimize the efficiency. Bisulfite-converted libraries were amplified using MyTaq Mix (Bioline) with the following program: 98°C for 2min, (98°C for 15sec, 60°C for 30sec, 72°C for 30sec) 12 cycles, 72°C for 5 min, 4°C indefinitely.
The library for sequencing was constructed using Ovation Ultralow DR Multiplex System 1-8 according to manufacturer's instructions (Nugen, San Carlos CA). Libraries were sequenced using HIseq-2000 (Illumina, San Diego, CA) to obtain 50 bp long reads.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Debarcoding of the multiplex runs was performed using in house shell script
RRBS data was aligned with bs-seeker2 (Guo et al., 2013).
Differentially methylated cytosines were calculated with methyl-kit (Akalin et al., 2012).
Genome_build: mm9
Supplementary_files_format_and_content: bigwig files
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| Submission date |
Mar 31, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Tõnis Org |
| E-mail(s) |
toniso@ut.ee
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| Phone |
372-52-11484
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| Organization name |
University of Tartu
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| Department |
Institute of Molecular and Cell Biology
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| Lab |
Biotechnology
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| Street address |
Riia 23
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| City |
Tartu |
| ZIP/Postal code |
51010 |
| Country |
Estonia |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE47085 |
Scl specifies hemogenic endothelium and inhibits cardiogenesis via primed enhancers |
| GSE56359 |
Scl specifies hemogenic endothelium and inhibits cardiogenesis via primed enhancers [RRBS] |
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| Relations |
| BioSample |
SAMN02712173 |
| SRA |
SRX504230 |
| Named Annotation |
GSM1359849_SclKO_MES_RRBS.bw |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1359849_SclKO_MES_RRBS.bw |
34.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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