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| Status |
Public on Sep 28, 2017 |
| Title |
RQ LL24 rep3 |
| Sample type |
SRA |
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| Source name |
aerial tissue harvested at LL24
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| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype: AtGRP7::AtGRP7 R49Q-GFP in grp7-1
age: 18 days
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| Treatment protocol |
Harvesting at timepoint LL24 and L36
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| Growth protocol |
Arabidopsis seeds from three genotypes (AtGRP7-GFP, AtGRP7-R49Q-GFP and GFP only) were surface-sterilized and sown on half-strength MS (Murashige-Skoog) (Duchefa) plates. Plants were grown in 12 h light/12 h dark cycles at 20 °C in Percival incubators (CLF laboratories) for 16 days followed by free run under continuous light (LL).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Seedlings on plates were subjected to irradiation with 254 nm UV light at a dose of 500 mJ/cm2 in a UVP CL-1000 UV crosslinker (on ice). Cell lysates from aerial tissue were prepared, precleared with sepharose beads and subjected to immunoprecipitation with GFP Trap beads (Chromotek) or mock precipitation with RFP Trap beads (Chromotek). On the beads, the precipitate was treated with DNase (Thermo Scientific). Subsequently, the RNAs were dephosphorylated and the L3 linker was ligated to the 3´ends using RNA ligase (NEB). 5′ termini were labeled using [γ-32P] ATP and polynucleotide kinase and the covalently linked RNA–protein complexes were separated on a 4-12 % NuPAGE Bis-Tris gel (Thermo Scientific), and electroblotted onto a nitrocellulose membrane. Upon autoradiography, the regions above the fusion protein were cut out and subjected to proteinase K treatment, leaving a polypeptide at the interaction site. Subsequently, RNA was isolated from the membrane using TriReagent.
RNA was reverse transcribed using primers containing a cleavable adapter region and individual barcode sequences. After NaOH treatment, the cDNA was purified on a 6% urea-polyacrylamide gel and fragments in the size range of approx. 70–85 nt (high, H), 85–120 nt (medium, M), and 120–200 nt (low, L), respectively, were eluted from the gel. The cDNAs were then circularized using CircLigase II (Epicentre) and an oligonucleotide (Cut-oligo) was annealed to generate a BamHI restriction site. Relineariztion via BamHI digestion results in adapters at both ends of the cDNA which then were PCR-amplified. After PCR optimization the three size fractions (H, M, L) are pooled with a ratio of 1:1:1, concentrations were assessed with Qubit dsDNA HS Assay Kit (Thermo Scientific).10 nM of the libraries were submitted to high throughput sequencing after multiplexing of multiple samples. Sequencing was carried out using an Illumina HiSeq2500 (Eurofins) with 50 nt single end or 100 nt single end reads.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Library strategy: iCLIP
Raw iCLIP reads were subjected to 3´ adapter trimming and quality filtering using cutadapt version 1.9.1
reads with a minimal length of 15 nt and a quality score of 20 were kept
The trimmed and filtered reads were de-multiplexed using a python script
Identical reads including the same random barcode sequence were considered PCR duplicates and hence removed
The barcodes were trimmed from the remaining reads using barcodeRemoval from PIPE-CLIP
The reads were mapped to the Arabidopsis thaliana TAIR10 reference genome with STAR v2.5.2a using the additional transcript annotation file atRTD.gff from atRTD allowing up to three mismatches. Only reads mapping uniquely were kept.
Crosslink sites (-1 position of the reads) were assigned to separate transcript regions; namely 5'UTR, Exon (concatenated), Intron and 3'UTR
significant crosslink sites determined by FDR (<0.05) as described in König et al. 2010, region wise
significant site determination was repeated 1000 times. Sites appearing at least 95% of repeats were kept.
Sites ocurring in most biological replicates were kept. (at LL24 2 of 3; 7GFP and GFP at LL36 4 of 5; RQ at LL36 2 of 2)
Genome_build: TAIR10
Supplementary_files_format_and_content: bedGraph files generated, representing significant crosslink sites. Score equals number of reads determined at the given position
Supplementary_files_format_and_content: significant_crosslinks.tar.gz (crosslink sites which were present in most replicates )
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| Submission date |
May 30, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Martin Lewinski |
| Organization name |
Bielefeld University
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| Department |
RNA Biology and Molecular Physiology
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| Street address |
Universitätsstraße 25
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| City |
Bielefeld |
| ZIP/Postal code |
33615 |
| Country |
Germany |
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| Platform ID |
GPL17639 |
| Series (2) |
| GSE99427 |
Identification of AtGRP7 targets by iCLIP |
| GSE99616 |
Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7 |
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| Relations |
| BioSample |
SAMN07177354 |
| SRA |
SRX2868788 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2643852_RQLL24rep3.bed.gz |
825 b |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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