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| Status |
Public on Apr 21, 2008 |
| Title |
rdd_RNA-seq |
| Sample type |
SRA |
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| Source name |
Immature floral tissue
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| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype/variation: ros1-3 dml2-1 dml3-1
tissue: unopened flower buds
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| Treatment protocol |
Immature (unopened) flower buds were collected and frozen in liquid nitrogen.
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| Growth protocol |
All plants were grown in potting soil (Metro Mix 250; Grace-Sierra, Boca Raton, FL) at 23C under a 16-hour light/8-hour dark cycle.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA-seq library construction protocol:
Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and treated with DNaseI (Qiagen) for 30 min at room temperature. following ethanol precipitation the 18S and 28S rRNA molecules were depleted from 20 µg of total RNA in three sequential Ribominus (Invitrogen, Carlsbad, CA) reactions as per manufacturer's instructions, using 6 plant-specific biotinylated LNA oligonucleotide rRNA probes supplied by (Invitrogen, Carlsbad, CA). The 5' cap was removed from the rRNA-depleted RNA by treatment with 10 U/µl Tobacco Acid Pyrophosphatase for 1.5 h at 37C. This and all subsequent enzymatic reactions involving RNA used contained 2.5-4 U/µl RNaseOut ribonuclease inhibitor (Invitrogen, Carlsbad, CA). The RNA was purified by phenol:chloroform extraction and ethanol precipitation. This and all subsequent ethanol precipitations contained 20-40 µg/ml nuclease-free glycogen (Ambion, Austin, TX). De-capped RNA was fragmented by metal hydrolysis in 1X fragmentation buffer (Affymetrix, Santa Clara, CA) for 35 min at 94 C then cooled on ice for 2 min and ethanol precipitated. The fragmented RNA was dephosphorylated using 10 U/µl Calf intestinal phosphatase (New England Biolabs, Cambridge, MA) for 1 h at 37C, then 10 µl Gel loading Buffer II (Ambion, Austin, TX) added, heated at 65'C for 5 min, cooled on ice and then separated on a 10% polyacrylamide gel containing 7 M urea in TBE buffer (45 mM Tris-borate, pH 8.0, and 1.0 mM EDTA) by electrophoresis at 150 V for 2 h at 4C. The gel was stained in SYBR Gold (Invitrogen, Carlsbad, CA), and a gel slice containing RNAs of 35 to 50 nucleotides was excised, crushed, and the RNA eluted in 0.3 M NaCl rotating at room temperature for 4 hours. The eluted RNAs were ethanol precipitated and resuspended in nuclease-free water, after which the RNA fragments were heated to 65'C for 5 min, cooled on ice for 5 min and then ligated to the Illumina 3' RNA oligonucleotide adapter (see smRNA library construction above) using 10 U/µl T4 RNA ligase (Promega, Madison, WI) in 10% DMSO, incubated at 20C for 6 h then 4 C for 4 h. Nucleic acids in the ligation reaction were separated by electrophoresis and a gel slice containing 3' adapter-ligated RNA molecules from 50 to 80 nucleotides was excised and the RNA eluted and precipitated as described above. The gel-purified RNA was resuspended in nuclease-free water then phosphorylated in a reaction containing 1 U/µl T4 polynucleotide kinase (New England Biolabs, Cambridge, MA) and 1 mM ATP (Illumina, San Diego, CA) for 1 h at 37 C. After purification by phenol:chloroform extraction and ethanol precipitation the 5' phosphorylated RNA fragments were ligated to the Illumina 5' RNA oligonucleotide adapter (see smRNA library construction above) under the same conditions used for the 3' adapter ligation. Nucleic acids in the ligation reaction were separated by electrophoresis and a gel slice containing 5' and 3' adapter-ligated RNA molecules from 80 to 125 nucleotides was excised and the RNA eluted as described above. The size-selected ligation products were then used as templates in a reverse transcription (RT) reaction, followed by a limited (20 cycle) PCR amplification step (see smRNA library construction above). The amplification products were separated by electrophoresis on a 6% polyacrylamide gel in TBE buffer and the 80 to 125 bp band excised. This cDNA was eluted in 1 X gel elution buffer (Illumina, San Diego, CA) rotating at room temperature for 3 hours, ethanol precipitated and resuspended in 15 µl elution buffer (Qiagen, Valencia, CA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
Sequence information was extracted from the image files with the Illumina Firecrest and Bustard applications and mapped to the Arabidopsis (Col-0) reference genome sequence (TAIR 7) with the Illumina ELAND algorithm. ELAND aligns 32 bases or shorter reads, allowing up to two mismatches to the reference sequence. For reads longer than 32 bases, only the first 32 bases will be used for alignment, while the remaining sequence will be appended regardless of similarity to the reference sequence. A Perl script was used to truncate the appended sequence at the point where the next four bases contain two or more errors relative to the reference sequence.
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| Data processing |
In order to avoid omitting unannotated transcripts, 36 nucleotide transcriptome reads were aligned to the Arabidopsis reference genome sequence (TAIR 7) with the ELAND algorithm.
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| Submission date |
Mar 26, 2008 |
| Last update date |
May 15, 2019 |
| Contact name |
Joseph R Ecker |
| E-mail(s) |
ecker@salk.edu
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| Phone |
8584534100
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| Organization name |
HHMI-Salk-Institute
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| Department |
Genomic Analysis Laboratory
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| Lab |
Ecker lab
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| Street address |
10010 North Torrey Pines Road
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL9062 |
| Series (2) |
| GSE10877 |
Highly integrated single base resolution maps of the epigenome in Arabidopsis |
| GSE10968 |
Highly integrated epigenome maps in Arabidopsis - transcriptome sequencing |
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| Relations |
| SRA |
SRX002557 |
| BioSample |
SAMN02195348 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM277615.txt.gz |
129.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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