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| Status |
Public on Nov 15, 2012 |
| Title |
col-0 ssRNA-seq |
| Sample type |
SRA |
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| Source name |
immature flower bud clusters
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| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: immature flower bud clusters
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| Extracted molecule |
total RNA |
| Extraction protocol |
The ssRNA-seq library was constructed as previously described (Li et al., 2012).
Ribo-seq libraries were made using ribosome-associated mRNAs from unopened flower buds that were isolated by differential centrifugation according to Mustroph et al. (Mustroph et al., 2009) with the following modifications. The ribosomes and associated mRNAs pelleted by centrifugation through a sucrose cushion were resuspended in 0.2 M Tris pH 8.0, 0.2 M KCl, 0.035 M MgCl2, 50 ug/ml chloramphenicol, and 50 ug/ml cycloheximide. Forty micrograms of resuspended RNA was centrifuged over a 15-60% sucrose gradient (0.04 M Tris, pH 8.0, 0.02 M KCl, 0.02 MgCl2, 5 ug/ml chloramphenicol, and 5 ug/ml cycloheximide). Following centrifugation, 50 ul fractions of the gradient were isolated and the OD260 of each was measured. The monosomal and polysomal fractions were pooled, and the RNA was isolated using the Qiagen miRNeasy Mini Kit. Eight micrograms of isolated RNA were depleted of ribosomal RNA using the RiboMinus Plant Kit (Life Technologies, Carlsbad, CA), fragmented using RNA Fragmentation Reagents (Ambion, Austin, TX), treated with T4 PNK (NEB, Boston, MA), and used for library preparation using the Illumina TruSeq smRNA-seq library preparation kit and accompanying protocols (Illumina, San Diego, CA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Treated with dsRNase (RNase ONE)
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| Data processing |
Basecalls performed with Bustard 1.13.48.
3' adapter sequences were trimmed using cutadapt (version 0.9.5 with parameters -e 0.06 -O 6 -m 14), and reads were subsequently aligned to the TAIR9 genome using bowtie (version 0.12.7 with parameters -n 1 -e 160 -l 25 -y -k 100 -B 1 –nomaqround).
Structure ‘hotspots’ and 'coldspots' were identified using a modified version of the CSAR software package (Muino et al., 2011). Specifically, structure scores were calculated for each base position in the genome and regions with significantly higher or lower than background at an FDR of 5% were called as ‘hotspots’ and ‘coldspots’, respectively. The background distribution was calculated by randomly shuffling dsRNA and ssRNA reads and then computing structure scores as described (Muino et al., 2011).
Genome_build: TAIR9
Supplementary_files_format_and_content: BED file: column 5 is [maximum absolute peak score]@@[location of peak score].
Supplementary_files_format_and_content: RPKM file is tab-delimited plain text format. Columns are TAIR ID, clone count, and RPKM.
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| Submission date |
Aug 17, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Fan Li |
| Organization name |
University of California Los Angeles
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| Department |
Pediatrics
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| Street address |
675 Charles E Young Drive South
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90024 |
| Country |
USA |
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| Platform ID |
GPL13222 |
| Series (1) |
| GSE40209 |
Regulatory impact of RNA secondary structure across the Arabidopsis thaliana transcriptome |
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| Relations |
| SRA |
SRX179273 |
| BioSample |
SAMN01120816 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM988391_col0_ssrna_hotspots_FDR05.bed.gz |
475.1 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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