 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Sep 28, 2017 |
| Title |
7GFP LL24 rep1 |
| Sample type |
SRA |
| |
|
| Source name |
aerial tissue
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype: AtGRP7::AtGRP7-GFP in grp7-1
|
| Treatment protocol |
Harvesting at timepoint LL24 and L36
|
| Growth protocol |
Plants grown in 12 h light-12 h dark cycles for 16 days and subsequently shifted to continuous light were vacuum-infiltrated with 1 % formaldehyde for 15 min at LL36 or LL24, followed by quenching with 125 mM glycine.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
A whole-cell extract was prepared in RIP-lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 4 mM MgCl2, 0.1 % Igepal, 1 % SDS, 5 mM DTT, 10 mM Vanadylribonucleosid complex, 100 U RiboLockTM/ml (Fermentas), 1 mM phenylmethylsulfonylfluorid and protease inhibitor tablets (Roche)). The extract was pre-cleared with Sepharose beads and subjected to immunoprecipitation with GFP-Trap® beads (Chromotek, Martinsried, Germany), hereafter called IP+. After extensive washing with RIP-washing buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 4 mM MgCl2, 5 mM DTT, 0.5 % Igepal, 1 % SDS, 0.5 % Na-deoxycholate, 2 M urea), coprecipitated RNAs were eluted with TriReagent and treated with DNase (Promega).
Libraries were prepared of three biological replicates using the Illumina TrueSeq Sample preparation kit v2, except for omitting the two rounds of poly(A) selection commonly used for total RNA as a starting material. Sequencing was carried out using an Illumina HiSeq2000 at the Genomics Center of the Max-Planck-Institute for Developmental Biology, Tuebingen, with 100 nt single-end reads.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
harvested at LL24
|
| Data processing |
Raw RIP-seq reads were subjected to quality trimming and filtering by Sickle v1.2 (https://github.com/najoshi/sickle) using parameters -l 50 -q 20.
The trimmed and filtered reads were mapped to the Arabidopsis thaliana transcriptome defined by atRTD.gff using STAR v2.5.2a with the parameter --quantMode TranscriptomeSAM
Estimated read counts per transcript were obtained by Salmon v0.8.2 and summarized into estimated read counts per gene by tximport
rRNA, mitochondrial and chloroplast genes were excluded from the analysis
Differentially expressed genes (DEGs) Enriched transcripts between RIP samples and RNA-seq samples were detected by edgeR as described in the tximport vignette (https://github.com/mikelove/tximport/blob/master/vignettes/tximport.md).
Genes with a FDR <0.001 and a log2-fold change > 0.5 were considered putative RIP targets
Genome_build: TAIR10
Supplementary_files_format_and_content: tab-delimited text file including TPM values in a matrix for each sample as columns
|
| |
|
| Submission date |
May 30, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Martin Lewinski |
| Organization name |
Bielefeld University
|
| Department |
RNA Biology and Molecular Physiology
|
| Street address |
Universitätsstraße 25
|
| City |
Bielefeld |
| ZIP/Postal code |
33615 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13222 |
| Series (2) |
| GSE99464 |
Identification of AtGRP7 targets by RIP-seq |
| GSE99616 |
Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7 |
|
| Relations |
| BioSample |
SAMN07178100 |
| SRA |
SRX2870766 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
