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Status |
Public on Nov 07, 2013 |
Title |
3T3 cells transfected with pCAG-EGFP_MosIR expressing long hairpin derived from mouse Mos gene |
Sample type |
SRA |
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Source name |
cell line
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Organism |
Mus musculus |
Characteristics |
cell line: NIH3T3 passages: 5-15 transfected plasmid: pCAG-EGFP_MosIR
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Treatment protocol |
NIH3T3 were co-transfected with pCAG-EGFP_MosIR and pcDNA4-DicerS-myc-His or pcDNA4-DicerS-myc-His (1.5 µg each per well in 6 well-plate). Cells were processed 48 hours post-transfection. Mouse embryonic stem cell lines were transfected with pCAG-EGFP_MosIR plasmid (3 µg per well in 6 well-plate). Cells were processed 48 hours post-transfection.
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Growth protocol |
3T3 cells were grown under standard tissue culture conditions in DMEM supplemented with 10% FCS. Mouse embryonic stem cells were cultured on gelatin-coated plastic in 2i-LIF medium: Knockout D-MEM (Invitrogen) containing 15% FCS (Sigma), 1× NEAA, 1 mM sodium pyruvate, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, supplemented with mouse LIF (1,000 U/mL), 3μM CHIR99021, and 1 μM PD0325901 (Sellect Chemicals).
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Extracted molecule |
total RNA |
Extraction protocol |
Samples processing and SOLiD sequencing was performed by Seqomics Ltd. (Hungary). Briefly, samples were depleted of ribosomal RNA using RiboMinus rRNA Removal Kit, eluted twice into 15 ul and treated with DNase I (5 ul 10x DNase I buffer, 2.5 U DNase I and water up to 50 ul reaction) 20 min at 37°C. Samples were ethanol-precipitated, eluted into 10 ul TE buffer and fragmented by RNase III treatment (1 ul 10x RNaseIII buffer, 8 ul sample, 1 ul RNaseIII) 10 min at 37°C. Fragmented samples were precipitated by ethanol and eluted into 4 ul. Adaptor was hybridized and ligated to each sample (10 ul 2x ligase buffer, 2 ul Adaptor mix, 3 ul hybridization solution, 2 ul ligation enzyme) by incubating O.N. at 16°C. Adaptor-ligated RNA was subjected to reverse transcription (4 ul 10x RT buffer, 2 ul dNTP mix, 2 ul RT primer, 1 ul ArrayScript RT, 13 ul water) for 40 min at 42°C. Resulting cDNA was purified using PCR Purification kit (Qiagen) and eluted into 10 ul. cDNA was run on 6% TBE-Urea gel and fragments 150-250 nt in length were excised. Isolated cDNA was amplified for 15 cycles using AmpliTaq DNA polymerase and purified by PCR Purification kit (elution twice in 10 ul). Second round of size selection on PAGE gel was performed and PCR products 110-130 nt in length were excised from gel and isolated. Purified amplicons were sequenced using ABI SOLiD 4 platform.
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Library strategy |
ncRNA-Seq |
Library source |
transcriptomic |
Library selection |
size fractionation |
Instrument model |
AB SOLiD 4 System |
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Data processing |
The given adapters were trimmed, and the raw files were filtered by quality using the cutadapt tool with the following command line: cutadapt -m 15 -e 0.1 -o fastq -c -z -a adapter csfastq csqual All fastq files were mapped in colorspace using the Shrimp short read aligner using following command line: shrimp -o 4 --strata -N 1 -L genome.index.i input.file The *.bed files were constructed using the bamToBed command from the bedtools toolkit. Genome_build: NCBI37/mm9 (without the chromosome containing "random" in their names) + pCAG-EGFP_MosIR plasmid sequence (available upon request). Supplementary_files_format_and_content: *.bed file format
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Submission date |
Sep 27, 2012 |
Last update date |
May 15, 2019 |
Contact name |
Radek Malik |
E-mail(s) |
malikr@img.cas.cz
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Organization name |
Institute of Molecular Genetics of the ASCR
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Department |
Dep. of Epigenetics Regulations
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Street address |
Videnska 1083
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City |
Prague 4 |
ZIP/Postal code |
CZ-14220 |
Country |
Czech Republic |
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Platform ID |
GPL14602 |
Series (1) |
GSE41207 |
A retrotransposon-driven Dicer variant enhances endogenous RNAi in mouse oocytes |
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Relations |
SRA |
SRX189909 |
BioSample |
SAMN01731161 |
Supplementary file |
Size |
Download |
File type/resource |
GSM1010538_3T3_MosIR.bed.gz |
322.0 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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