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Status |
Public on Mar 01, 2012 |
Title |
rdr6-CMV∆2b_rep1_At_matches |
Sample type |
SRA |
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Source name |
Systemically-infected leaves
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Organism |
Arabidopsis thaliana |
Characteristics |
genotype: rdr6-15 genotype reference: Wang et al., 2010. PNAS 107(1):484-9. source: Total RNA
|
Growth protocol |
Arabidopsis plants were grown in our glass houses under stardard conditions.
|
Extracted molecule |
total RNA |
Extraction protocol |
Low molecular weight RNAs extracted by Trizol (Invitrogen) from the systemically infected leaves 14 days after inoculation with CMV-Δ2b were purified from 15% denatured polyacrylamide gel. Purified small RNAs were directly ligated to the 3′-linkers for miRNA cloning (Integrated DNA Technology) by T4 RNA ligase 2Tr (New England Biolabs). The ligation products were purified and ligated to a suitable 5′ Solexa linker by T4 RNA ligase I (New England Biolabs). The final ligation products were reverse transcribed using a primer targeting the 3′-linker and the cDNA pool was amplified by PCR and sequenced by the Solexa technology (Illumina) in our campus facility. Duplicate libraries were constructed from two independent RNA samples. immunoprecipitation: Three g systemic leaves from FLAG-AGO1 or HA-AGO2 plants infected with CMV-Δ2b were ground to powder in liquid nitrogen and supplied with 10 ml of extraction buffer [20 mM Tris•HCl, pH 7.5/300 mM NaCl/5 mM MgCl2/5 mM DTT/1% plant protease inhibitor mixture (Sigma)]. The suspension was clarified by centrifugation (10 min, 4ºC, 14,000 rpm) and the supernatant filtered through cheesecloth. The filtered supernatant was incubated with anti-FLAG M2 agarose (Sigma-Aldrich) or anti-HA affinity matrix (Roche), according to manufacturers’ protocols, for 4h at 4ºC. The beads were then washed twice with extraction buffer supplemented with 0.5% Nonidet P-40 and the AGO-bound RNA extracted with TRIzol.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
size fractionation |
Instrument model |
Illumina Genome Analyzer |
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Data processing |
Reads were extracted using the Illumina pipeline. Trimming of adapters and down-stream analyses were carried out with in-house Perl scripts, except alignments of short reads to Arabidopsis or CMV sequences, which were performed with bowtie (no mistmatches were allowed). All sequences containing perfect match 5' barcode (ATC/CGT/TAC) and at least 6 sucessive perfect-matched nucleotides of the 3' linker were kept for later analysis. The barcode and linker sequences were computationally removed by in-house perl scripts. All trimmed sequences in length of 18-30nt were aligned with CMV genome and the perfect matched reads were used for all further analysis.
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Submission date |
Feb 23, 2011 |
Last update date |
Jun 11, 2013 |
Contact name |
JUAN JOVEL |
E-mail(s) |
jjovel@catie.ac.cr
|
Organization name |
University of California Riverside
|
Department |
Plant Pathology
|
Lab |
Ding
|
Street address |
3401 Watkins Drive
|
City |
Riverside |
State/province |
CA |
ZIP/Postal code |
92521 |
Country |
USA |
|
|
Platform ID |
GPL9062 |
Series (1) |
GSE27477 |
Small RNAs from Arabidopsis plants infected with CMV-∆2b |
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Relations |
BioSample |
SAMN02197010 |