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| Status |
Public on May 17, 2016 |
| Title |
TTP PAR-iCLIP rep1 |
| Sample type |
SRA |
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| Source name |
TTP PAR-iCLIP
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: Primary bone marrow derived macrophages
clip antibody: anti-TTP
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| Treatment protocol |
BMDMs were cultured on 15 cm cell-culture treated dishes in medium supplemented with 100 µM 4-thiouridine (Sigma) for 16 hours prior to experiment. The cells were then stimulated with LPS (10 ng/ml, Sigma) for 6 hours.
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| Growth protocol |
Murine bone marrow derived macrophages were produced from the bone marrow isolated from femur and tibia of 7- to 9-week-old TTPΔM or WT littermates’ mice. Macrophages were cultivated in DMEM (Sigma) supplemented with 10% FBS (Sigma), 100 U/ml penicillin (SIgma), 100 μg/ml streptomycin (Sigma) and CSF-1 derived from L929-cells as previously described (Kovarik et al. 1998).
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| Extracted molecule |
total RNA |
| Extraction protocol |
As described at Konig J, Zarnack K, Rot G, Curk T, Kayikci M, Zupan B, Turner DJ, Luscombe NM, Ule J. 2010. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol 17(7): 909-915; Hafner M, Landthaler M, Burger L, Khorshid M, Hausser J, Berninger P, Rothballer A, Ascano M, Jr., Jungkamp AC, Munschauer M et al. 2010. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141(1): 129-141.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
TTP bound RNA
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| Data processing |
Reads were mapped to mouse genome (mm9 assembly) with Segemehl v0.1.3-349M
Peak finding was performed using Pyicos ModFDR method for peak finding, together with a modified filtering algorithm for the use with PAR-iCLIP crosslink sites, which can be seen as reads of length one.
Peak filtering: Our custom filtering method splits peak regions surrounding the highest peak signal, henceforth named summit in accordance with Pyicos, once certain height-thresholds are reached. Cutoffs were defined based on signals detected in known TTP targets. Peaks with a summit signal below 100 pileups are considered background and discarded. With a sliding window approach, starting from the summit, a peak is first split when its height falls below 30% of the summit signal. Emerging subpeaks with a summit above this cutoff and 100 pileups are then recursively split when their signal falls below 10% of their summit. Final split-peaks contain a high amount of crosslink signal and allow us to analyze protein binding sites with high resolution. Replicates of each experimental setup were analyzed separately. Width and position of peaks vary slightly between experiments. For the ranked lists of TTP and HuR target genes, we collect peaks from all replicates and filter out peaks that do not have an overlap with peaks in all other replicates.
Genome_build: mm9
Supplementary_files_format_and_content: tables include annotation of filtered peaks in the following format chromosome:start-end(strand), peak score, genomic feature (5' UTR, CDS, 3' UTR, intron)
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| Submission date |
Nov 19, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Vitaly Sedlyarov |
| Phone |
+4314016070050
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| Organization name |
Research Center for Molecular Medicine
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| Lab |
Prof. Giulio Superti-Furga
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| Street address |
Lazarettgasse 14, AKH BT 25.3
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| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE63466 |
Pervasive TTP binding but selective target mRNA destabilization in the macrophage transcriptome [PAR-iCLIP] |
| GSE63468 |
TTP binding site atlas in the macrophage transcriptome reveals a switch for inflammation resolution |
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| Relations |
| BioSample |
SAMN03201425 |
| SRA |
SRX763879 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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