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| Status |
Public on Feb 11, 2013 |
| Title |
SB |
| Sample type |
SRA |
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| Source name |
TAG 1 ES_SB
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| Organism |
Mus musculus |
| Characteristics |
cell type: TAG 1 ES cells
treated with: SB-431541 treated (P-Smad2/3 repressed)
molecule subtype: Total RNA containing short RNA fraction
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| Treatment protocol |
RNA was extracted from mouse ES cells which were either untreated (control sample), treated for 16 hrs with 30 µM SB-431541 (SB sample), or for 16 hrs 30 µM SB followed by 12 hrs 1 µg/µl Dox (Dox sample)
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| Growth protocol |
ES cells were were maintained in DMEM (Invitrogen) containing 15% Foetal Bovine Serum (FBS) (Source Bioscience) and Leukemia Inhibitory Factor (LIF) (homemade), 2 mM L-Glutamine, 1% Penicillin-Streptomycin, 0.1 nM b-Mercaptoethanol at 37°C in a 5% CO2 humidified incubator
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA containing the short RNA fraction was isolated using the miRVana miRNA isolation kit (Ambion). Libraries were prepared according to the Illumina small RNA preparation protocol v1.0. The 20-30 nucleotide fraction was isolated from 10µg of total RNA after being run in a 15% Urea-TBE gel (Invitogen) for 1h. The RNA contained on the excised gel bands was eluted on 300μl of 0.3M NaCl solution during 4h at RT and constant rotation. The elute was separated from the gel debris through a Spin-X-column (Fisher) and RNA was precipitated by adding 750μl of 100% ethanol and 3μl of glycogen (Ambion) (1mg/ml) and incubating it for 30min at -80˚C. The precipitated RNA was centrifuged at 14K rpm for 25 min at 4˚C, washed with 75% ethanol and resuspended on 5.7μl of RNAse free water. A 5’ adaptor was ligated to the RNA in a reaction containing the full amount of RNA recovered from last step, 1.3μl of SRA 5’ adaptor (Illumina), 1μl T4 RNA ligase (10U/μl) (Promega), 1 μl 10X T4 RNA ligase (Promega) and 1 μl RNAseOUT (Invitrogen). The reaction was incubated at 20˚C in a thermal cycler for 6h. The resulting product was run on a 15% Urea-TBE gel (Invitrogen) and the band corresponding to 40-60 nucleotides was excised. The 5’ ligated RNA was eluted and precipitated as described before and finally resuspended in 6.4 μl of RNAse free water. A 3’ adaptor was added to these molecules in a reaction containing the entire 5’ ligated RNA recovered from last step, 0.6 μl of SRA 3’ adaptor v 1.0 (Illumina), 1μl T4 RNA ligase (10U/μl) (Promega), 1 μl 10X T4 RNA ligase (Promega) and 1 μl RNAseOUT (Invitrogen). The reaction was incubated at 20˚C in a thermal cycler for 6h. The product obtained was run on a 10% Urea-TBE gel (Invitrogen) and the band corresponding to 70-90 nucleotides was excised. The 5’ and 3’ ligated RNA was eluted and precipitated as described before and finally resuspended in 4.5 μl of RNAse free water. In order to synthesize single stranded DNA from this material, 0.5 μl of SRA RT-primer (Illumina) were added to it and the mixture was incubated at 65˚C for 10 min. This was complemented with 2 μl of 5X first strand buffer, 1 μl 100mM DTT, 0.5 μl RNAseOUT (all from Superscript II reverse transcription kit (Invitrogen)) and 0.5 μl 12.5mM dNTP mix (BioLine) and incubated at 48˚C for 3 min. After adding 1 μl of Superscript II retrotranscriptase (Invitrogen), the reaction was incubated at 44˚C for 1h. A PCR reaction was then set up with the resultant product and 0.5µl GX1 primer, 0.5µl GX2 primer (both from Illumina), 0.5µl 25mM dNTP mix (Bioline), 10µl 5X cloned Phu buffer, 0.5µl Phu polymerase (both from NEB) and 28µl RNAse free water. The cycling conditions were as follows: 1 cycle of 98˚C for 30 sec; 15 cycles of 98˚C for 10 sec, 60˚C for 30 sec and 72˚C for 15 sec; 1 cycle of 72˚C for 10 min. The PCR product was purified by running it on a 10% TBE-PAGE gel for 35 min. The band around 90bp was excised and eluted on 100µl of 1X Elution buffer 2 (NEB) for 2h at RT and at constant rotation. The elute was separated from the gel debris through a Spin-X-column (Fisher) and DNA was precipitated by adding 1μl of glycogen (1mg/ml) (Ambion), 10ul 3M sodium acetate (Ambion) and 325μl of -20˚C100% ethanol and centrifuging at 14K rpm for 20 min. After a wash with 70% ethanol, the DNA was vacuum dried and resuspended in 10µl of water.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Sample 2
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| Data processing |
Two lanes of single-end sequencing reads of 36bp were generated from each of the three libraries in a GAII Illumina Genome Analyser. The sequencing reads for each sample were combined and trimmed from the adaptor using the FASTQ/A Clipper from the FASTX tool kit (http://hannonlab.cshl.edu/fastx_toolkit/). Only reads with a length greater than 16 bases were selected for mapping.
Bowtie (version 0.12.7) (http://bowtie-bio.sourceforge.net/index.shtml) was used to align the selected reads to the miRNA mature sequences for Mus musculus included in the mature.fa file from miRBase (version 18) (http://www.mirbase.org/). In order to allow only perfect matches, the default parameters were used except for the following: “—seedmms 0 --seedlen 60 --maqerr 10 -m 2000000 -k 2000000”. Reads mapping to a unique position were assigned to the particular miRNA. Reads mapping to several positions were assigned to the miRNA with the same length whenever possible or to the miRNA with the closest length. In case that more than one miRNA fulfilled this requirement the read was then assigned to each of these miRNAs.
Reads resulting unmmaped to mature forms were subsequently aligned to the miRNA precursor sequences for Mus musculus included in the hairpin.fa from miRBase (version 18) by using the same parameters described before. In order to assign the aligned reads to mature forms produced from the matching precursor, the coordinates for all mature forms derived from the 5p and the 3p arms of all miRNA precursors were determined as follows: mature forms for Mus musculus extracted from the mature.fa file from miRBase (version 18) were mapped to the precursor sequences (Mus musculus haipin.fa, miRBase, version 18) using Bowtie and the same parameters described previously. These coordinates were used to assign the reads mapped to precursors to the corresponding mature form derived from these precursors. In order for a read to be assigned to a certain mature form, total or partial overlap to the specified coordinates was required. Under these conditions, reads mapping to one position were assigned to the respective mature miRNA. Since some mature forms can be produced from more than one precursor, reads mapping across all precursors were identified and assigned only once to the corresponding mature miRNA. For any remaining reads mapping to several positions in the hairpin.fa, all positions were accepted.
We only considered a miRNA to be expressed if 10 or more reads were found across all 3 samples.
Genome_build: miRBase (mouse, version 18)
Supplementary_files_format_and_content: Tab-delimited text files include reads normalized to the total number of reads for the respective sample and multiplied by 1,000,000 in order to scale the values to the range of "reads per million".
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| Submission date |
Aug 08, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Carme Camps |
| Organization name |
Wellcome Trust Centre for Human Genetics/ U. of Oxford
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| Street address |
Roosevelt Drive
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| City |
Oxford |
| ZIP/Postal code |
OX37BN |
| Country |
United Kingdom |
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| Platform ID |
GPL9250 |
| Series (1) |
| GSE39994 |
TGF-beta/Smad2/3 signaling directly regulates several miRNAs in mouse ES Cells and early embryos |
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| Relations |
| SRA |
SRX174807 |
| BioSample |
SAMN01109514 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM983068_SB_normalised_miRNA_reads.txt.gz |
5.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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