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| Status |
Public on Apr 27, 2012 |
| Title |
TNGA-serum_strand-specific-rRNA-depleted-RNA-Seq |
| Sample type |
SRA |
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| Source name |
Embryonic Stem cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6 * 129
cell type: ES-cells
source: ds_cDNA
antibody: rRNA depleted strand specific ds_cDNA
rna subtype: rRNA depleted total RNA
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| Treatment protocol |
No specific treatments
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| Growth protocol |
Embryonic stem cells were cultured without feeders in the presence of leukaemia inhibitory factor (LIF) either in medium containing 10% foetal calf serum or in serum-free medium containing the MEK inhibitor PD0325901 (1μM) and the Gsk3 inhibitor CH99021 (3μM), together known as 2i. TNGA female ES cells on a mixed strain 129 and C57BL/6 background and female and male NOD ES cells from the non-obese diabetic strain were derived in 2i. TNGA cells are heterozygous for knock-in of eGFP to the Nanog gene. The female XT67E1 line originates from a mixed 129 and PGK/C3H background, and contains a monoallelic deletion in the Xist gene. E14Tg2a, RGD2, Rex-GFP and HM1 cells are male ES cells of 129 background, established and grown in serum without feeders according to standard protocols. Rex-GFP cells contain a monoallelic GFP knock-in substitution at the endogenous Rex1 locus.
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| Extracted molecule |
total RNA |
| Extraction protocol |
ChIP experiments were performed using 3.3x106 cells and 3µg antibody per ChIP according to standard protocols as described with two minor modifications. Crosslinking of the cells was performed on the culture plates for 20 minutes, while ChIP’ed DNA was purified by Qiaquick PCR purification Kit (Qiagen). ChIP enrichment levels were analyzed by qPCR for quality control. Total RNA was isolated using Trizol (Invitrogen) and subjected to chloroform extraction, isopropanol precipitation and ethanol washing according to the manufacturer's recommendations. 100µg total RNA was subjected to two rounds of poly(A) selection using the Oligotex mRNA Mini Kit (Qiagen), followed by DNaseI treatment (Qiagen). 100-200ng 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 seconds and purified using the RNAeasy Minelute Kit (Qiagen). The fragmented mRNA was used as template for cDNA synthesis using 5µg random hexamers using Superscript III Reverse Transcriptase (Invitrogen) according to the manufacturer's recommendations. Ds-cDNA was synthesized in second strand buffer (Invitrogen) according to the manufacturer's recommendations and purified using the Minelute Reaction Cleanup Kit (Qiagen). Strand specific rRNA depleted ds_cDNA profiling was performed using the ScriptSeq kit (cat. no. SS10924) from Illumina, according to the instructions of the manufacturer. rRNA depletion was performed using the Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat; cat. no. RZH110424). Quality control was performed by qPCR.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
ChIP DNA or ds-cDNA samples were prepared for sequencing by end repair of 20 ng DNA as measured by Qubit (Invitrogen). Adaptors were ligated to DNA fragments, which were subsequently size selected (~300bp). The adapter-modified DNA fragments were subjected to limited PCR amplification (14 cycles). Quality control was made by qPCR and by running the products on a Bioanalyzer (BioRad). Finally, cluster generation and sequencing-by-synthesis (36 bp) was performed using the Illumina Genome Analyzer IIx (GAIIx) platform according to standard protocols of the manufacturer (Illumina). The image files generated by the Genome Analyzer were processed to extract DNA sequence data. Sequences were aligned to the mouse MM9 reference genome using the Illumina Analysis Pipeline allowing one mismatch. Samples were sequenced to a depth of approximately 10-20 million mapped tags per sample (Table S1). Unless specified otherwise, only the tags uniquely aligning to the genome were considered for further analysis. For RNA-Seq, further analysis was performed using the 36 bp sequence reads. For ChIP-Seq, the 36 bp sequence reads were directionally extended to 300 bp, corresponding to the length of the original fragments used for sequencing, and tags mapping on exact the same genomic locus were discarded to obtain a non-redundant set. The output data were converted to Browser Extensible Data (BED) files for downstream analysis and Wiggle (WIG) files for viewing the data in the UCSC Genome Browser. All ChIP-Seq and RNA-Seq sequence analyses were conducted based on the Mus musculus NCBI m37 genome assembly (MM9) accessed from the Ensembl (release 54; May 2009) or the UCSC (assembly July 2007) Genome Browsers.
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| Submission date |
Dec 16, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Hendrik Marks |
| E-mail(s) |
h.marks@ncmls.ru.nl
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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 |
GPL11002 |
| Series (1) |
| GSE23943 |
Epigenome and transcriptome of naive pluripotent mouse embryonic stem (ES) cells cultured in 2i serum-free medium |
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| Relations |
| SRA |
SRX112911 |
| BioSample |
SAMN00768076 |
| Named Annotation |
GSM850401.bed.gz |
| Named Annotation |
GSM850401.wig.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM850401.bed.gz |
110.0 Mb |
(ftp)(http) |
BED |
| GSM850401.wig.gz |
24.2 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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