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| Status |
Public on Apr 28, 2014 |
| Title |
EpiSC_1_FT_8.73_p19_RNA_Seq |
| Sample type |
SRA |
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| Source name |
EpiStem Cells (EpiSC)
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| Organism |
Mus musculus |
| Characteristics |
cell type: EpiSC
measurement: RNA
passage: 19
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| Growth protocol |
EpiSCs and cEpiSCs were grown in serum-free medium (CDM) with FGF2 (12ng/mL, R&D) and Activin A (20ng/ml, R&D) on serum coated dishes as described (Brons et al., 2007).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Genomic DNA was purified using DNA extraction kit (Promega) according to the manufacturer’s instructions. DNA was extracted pools of seven E6.5 epiblasts or pools of three E7 epiblasts. After proteinase K digestion in lysis saline buffer DNA was extracted using NaCl/EtOH precipitation.Genomic DNA was sonicated to generate 300 bp fragments on average. MethylCap was performed using the IP-STAR robot (Diagenode) as described before (Bock et al., 2010). In short, 1 μg DNA was incubated with paramagnetic beads coated with the MBD domain of MeCP2 fused to GST. After washing with 200 mM, 400 mM and 500 mM NaCl, the bound methylated DNA was eluted in two fractions using 600 mM and 800 mM NaCl, respectively. 20 ng of DNA eluates was prepared for sequencing. For RNA-Seq, Total RNA was isolated with Trizol (Invitrogen) according to the manufacturer’s recommendations. 100 μg total RNA was subjected to two rounds of poly(A) selection (Oligotex mRNA Mini Kit; QIAGEN), followed by DNaseI treatment (QIAGEN). 100–200 ng mRNA was fragmented by hydrolysis (5x fragmentation buffer: 200mM Tris acetate, pH8.2, 500mM potassium acetate and 150mM magnesium acetate) at 94°C for 90 s and purified (RNAeasy Minelute Kit; QIAGEN). cDNA was synthesized using 5 μg random hexamers by Superscript III Reverse Transcriptase (Invitrogen). Double-strand cDNA synthesis was performed in second strand buffer (Invitrogen) according to the manufacturer’s recommendations and purified (Minelute Reaction Cleanup Kit; QIAGEN). 20 ng was used for sequencing.
DNA or cDNA samples were prepared for sequencing by end repair of 20 ng DNA as measured by Qubit (Invitrogen). Adaptors were ligated to DNA fragments, followed by size selection (~300 bp) and 14 cycles of PCR amplification. Integrity of DNA libraries was confirmed by running the products on a Bioanalyzer (BioRad). Cluster generation and sequencing (36 bp) was performed with the Illumina Genome Analyzer IIx (GAIIx) (MethylCap-seq) or HiSeq (RNA-seq) platform according to standard Illumina protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Initial data processing, base calling, and alignment to the mouse reference genome was performed using the Illumina Analysis Pipeline allowing one mismatch. Only tags aligning to one position on the genome were considered for further analysis.
For RNA-seq, further analysis was performed with the 36 bp aligned sequence.
For MethylCap-seq, the uniquely mapped sequence reads were directionally extended to 300 bp, the estimated median length of the original DNA library. If multiple tags mapped on the same genomic position, only one was included for further analysis. Mapped reads from the initial 600 mM and 800 mM NaCl eluate libraries were combined.
For both RNA-seq and MethylCap-seq data was converted to Browser Extensible Data (BED) files for downstream analysis. To compensate for differences in sequencing depth and mapping efficiency among samples, the total number of unique reads of each sample was uniformly equalized relative to the sample with the lowest number of sequence reads, allowing quantitative comparisons.
Wiggle (WIG) files for viewing the data in the UCSC Genome Browser were generated from the normalized files.
Genome_build: mm9
Supplementary_files_format_and_content: Bed files were generated according to general instructions and were used for analysis; Wig files can be directly uploaded to the UCSC genome browser.
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| Submission date |
Jun 10, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Hendrik Marks |
| E-mail(s) |
[email protected]
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| Organization name |
Radboud University Nijmegen, RIMLS
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| Department |
Molecular Biology
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| Street address |
Geert Grooteplein 26/28
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| City |
Nijmegen |
| ZIP/Postal code |
6525GA |
| Country |
Netherlands |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE47793 |
The DNA methylome of Epiblast Stem Cells and Embryonic Stem Cells is distinct and not fully reversible |
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| Relations |
| BioSample |
SAMN02194178 |
| SRA |
SRX298377 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1159713_EpiSC_1_FT_8.73_p19_RNA_Seq.bed.gz |
73.3 Mb |
(ftp)(http) |
BED |
| GSM1159713_EpiSC_1_FT_8.73_p19_RNA_Seq.wig.gz |
9.7 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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