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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 31, 2019 |
| Title |
mESC 0h chase Rep 2 |
| Sample type |
SRA |
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| Source name |
mouse embryonic stem cells AN3-12
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| Organism |
Mus musculus |
| Characteristics |
treatment: 12h s4U treatment
genotype: AN3-12
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| Treatment protocol |
Mouse embryonic stem cells were incubated with 100 µM s4U for 12h; followed by a wash out with incubation with standard medium supplemented with 10 mM uridine for the times indicated before they were directly lysed in Trizol® (Ambion).
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| Growth protocol |
Mouse embryonic stem (mES) cells (clone AN3-12) were cultured in 15 % FBS (Gibco), 1x Penicillin-Streptomycin solution (100 U/ml Penicillin, 0.1 mg/ml Streptomycin, Sigma), 2 mM L-Glutamine (Sigma), 1x MEM Non-essential amino acid solution (Sigma), 1 mM sodium pyruvate (Sigma), 50 µM 2-Mercaptoethanol (Gibco) and 20 ng/ml LIF (in-house produced). Cells were maintained at 37°C with 5% CO2 and passaged every second day.
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| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA extraction and IAA-derivatization
Targeted mRNA 3' end cDNA libraries were prepared using a protocol described in the Quantseq Flex kit. For details see associated manuscript. Gene-specific forward primers were as follows (sequence in 5 -3 ): ActB,ENSMUSG00000029580, CACGACGCTCTTCCGATCTNNNNNNCCCACTCCTAAGAGGAGGATG; GusB, ENSMUSG00000025534, CACGACGCTCTTCCGATCTNNNNNNGTGGTCTTTGGGAAGGTGAA; Hspa1b, ENSMUSG00000090877, CACGACGCTCTTCCGATCTNNNNNNGGACTGGATGTTCCTCTCCA; Nat10, ENSMUSG00000027185, CACGACGCTCTTCCGATCTNNNNNNTGAAGGGTGGTGTTTTCTCA; Sox2, ENSMUSG00000074637, CACGACGCTCTTCCGATCTNNNNNNATGGCCAACTTTGGCAGTAG; Tnrc6b, ENSMUSG00000047888, CACGACGCTCTTCCGATCTNNNNNNTTTTAGGGGCATCACTGGAG;
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Description |
artificial_chromosome_targetedRNAseq.bed
artificial_chromosome_targetedRNAseq.fa
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| Data processing |
Basecalls performed using "Real Time Analysis RTA" V.1.18.64.0
Demultiplexed reads were adapter-trimmed using cutadapt; 12nt were trimmed from the 5' during the alignment process
Trimmed reads were aligned using SLAMdunk v0.3.0: Reads were aligned to the artificial chromosome (artificial_chromosome_targetedRNAseq.fa) using local alignment scoring and multimapping reads were discarded. In the filtering step, alignments with a minimum identity of 95% and a minimum of 50% of the read bases mapped were retained. SNPs exceeding a coverage cutoff of 10x and a variant fraction cutoff of 0.8 were called using VarScan2.4.1 using default parameters. Non-SNP overlapping T>C mutations with a base quality of Phred score >26 were identified. T>C containing reads and total reads aligning within the custom defined amplicon regions (artificial_chromosome_targetedRNAseq.bed) were reported. T>C conversion rate was determined for each position along the custom defined intervals by normalizing to genomic T content and coverage of each position and averaged per region.
The artifical_chromosome_targetedRNAseq.bed file was manually created by extracting the genome coordinates and sequences (for fasta file) of the expected amplicons of the custom-designed primers
Genome_build: artificial_chromosome_targetedRNAseq.fa
Supplementary_files_format_and_content: artificial_chromosome_targetedRNAseq.fa: Genomic sequences of the expected amplicons of the custom-designed primers. artificial_chromosome_targetedRNAseq.bed: Relative Coordinates of the amplicons of the custom-designed primers. *.tsv files: Count file output from SLAMdunk containing the relative chromosomal coordinates (from the "artificial chromosome"), Gene Name and Strand of the sequenced amplicons, T>C conversion rate, Expression (ReadsCPM), T content of the amplicon interval, Coverage on Ts within the amplicon interval, T>C Conversions on Ts, Read Count, T>C Read Count and Multimap Counts
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| Submission date |
Aug 03, 2018 |
| Last update date |
Jan 01, 2020 |
| Contact name |
Brian Reichholf |
| E-mail(s) |
brian.reichholf@imba.oeaw.ac.at
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| Organization name |
IMBA
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| Lab |
Stefan Ameres
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| Street address |
Doktor-Bohr-Gasse 3
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| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platform ID |
GPL16417 |
| Series (1) |
| GSE118121 |
Chemical nucleoside conversion-based measurement of RNA stability by targeted and untargeted mRNA 3´ end sequencing |
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| Relations |
| BioSample |
SAMN09765546 |
| SRA |
SRX4507294 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3318876_70343_mESC_0h_chase_R2_reduced.tsv.gz |
440 b |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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