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Sample GSM678877 Query DataSets for GSM678877
Status Public on Mar 01, 2012
Title rdr6-CMV∆2b_rep1_At_matches
Sample type SRA
 
Source name Systemically-infected leaves
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection size fractionation
Instrument model Illumina Genome Analyzer
 
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.
 
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
Relations
BioSample SAMN02197010

Supplementary file Size Download File type/resource
GSM678877_rdr6-CMVd2b_rep1_At_matches.txt.gz 4.0 Mb (ftp)(http) TXT
Processed data provided as supplementary file

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