NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM1359848 Query DataSets for GSM1359848
Status Public on Jan 01, 2015
Title WT_MES_RRBS
Sample type SRA
 
Source name ES cell derived day4 EB (embryoid body) Flk1+ mesodermal cells
Organism Mus musculus
Characteristics cell-line: WT J1 mESC
cell type: WT ES cell derived day4 EB (embryoid body) Flk1+ mesodermal cells
cell-line: WT J1 mESC
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.
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.
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.
 
Library strategy Bisulfite-Seq
Library source genomic
Library selection Reduced Representation
Instrument model Illumina HiSeq 2000
 
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
 
Submission date Mar 31, 2014
Last update date May 15, 2019
Contact name Tõnis Org
E-mail(s) toniso@ut.ee
Phone 372-52-11484
Organization name University of Tartu
Department Institute of Molecular and Cell Biology
Lab Biotechnology
Street address Riia 23
City Tartu
ZIP/Postal code 51010
Country Estonia
 
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]
Relations
BioSample SAMN02712186
SRA SRX504229
Named Annotation GSM1359848_WT_MES_RRBS.bw

Supplementary file Size Download File type/resource
GSM1359848_WT_MES_RRBS.bw 37.1 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap