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Sample GSM2337661 Query DataSets for GSM2337661
Status Public on Jun 12, 2017
Title Stage 1 uninfected
Sample type SRA
Source name ROOT
Organism Solanum lycopersicum
Characteristics cultivar: Pusa ruby
tissue: root
age: 5 week old + (1dpi+2dpi+3dpi)
uninfected or infected: uninfected
Treatment protocol The second juvenile stage (J2) is the only parasitic stage that was used for infecting plants, therefore, egg masses were detached manually from infected roots and kept in deionised water at room temperature for hatching of eggs into J2s. After 2-3 days, J2s were separated from unhatched eggs by passing the suspension of J2s through three to four layered kim wipes. The five-week old plantlets of both susceptible cv. and resistant line were grown in sterile soil and inoculated with 1500 J2s. The control plantlets were also raised that have not been exposed to nematode. For tissue collection, in susceptible cv. for 30 days and resistant line for 7 days, plantlets were uprooted, roots were quickly washed under cold tap water, excised from the plant and snap chilled in liquid nitrogen. Tissue was kept in -80o C for subsequent storage. The root tissue of uninfected plantlets of the same age was also harvested as control samples. The additional one plant was harvested on each day to study the lifecycle of nematode inside the root and progression of the disease through acid fuchsin staining (Bybd et al. 1983). After uprooting, roots were washed and suspended in 1.5% sodium hypochlorite bleach for 4 mins and then washed to remove excess bleach. For staining, roots were boiled for a minute in 0.2% acid fuchsin stain prepared in lactophenol followed by clearing of roots. The whole plant root system was observed under the inverted microscope (Carl Zeiss, Gottingen, Germany) and number of nematodes at their different development stage were counted. Five disease development stages of susceptible response and two disease development stages of resistance response were selected for further study on the basis of the rate of nematode invasion, a number of knots/galls formed on infected root, transition of varied developmental stages of nematode and progression of disease in the host.
Growth protocol Disease development study was conducted using root knot nematode, M. incognita. RKN was propagated on susceptible Indian tomato cv. Pusa Ruby in infection house at Department of Botany, University of Delhi, India. Infection studies were conducted under controlled growth conditions at 22oC with 16 hour light and 8 hour dark photoperiod. For miRNA studies, tomato susceptible cv., Pusa Ruby and tomato resistant transgenic line that contains Mi-1 resistance gene were used.
Extracted molecule total RNA
Extraction protocol Root tissue of equal weight of each disease development stage was processed for RNA isolation using TRI reagent (Sigma-Aldrich, St. Louis, MO, U.S.A.) as per manufacturer’s instructions. The integrity of RNA was checked on 1.2% agarose gel and quantification was done through optical density measurement using nanovue (GE Healthcare Bio-sciences, Uppsala, Sweden).
In total 11 small RNA libraries were prepared including five infected disease development stages and their corresponding uninfected tissue of same age and a library of five-week old uninfected root tissue of susceptible tomato cv. Small RNA libraries from root tissue of each stage were prepared independently using Illumina small RNA sample preparation kit (Illumina, San Diego, CA, U.S.A.) according to manufacturer’s instructions. Briefly, an equal amount of total RNA of each stage was used for sequential ligation of 3’and 5’adapter using T4 RNA ligase. Adapter ligated small RNA was reverse transcribed using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA, U.S.A.) and 3’ adapter specific RT-primer. cDNA prepared was amplified and product of approximately 147 bp was eluted through 6% polyacrylamide gel. The quality and quantity of cDNA libraries prepared were determined through Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, U.S.A.). High-throughput sequencing of libraries was performed using Illumina Hiseq 2000 system at Institute of Genomics and Integrative Biology, Delhi, India, according to manufacturer’s instructions.
Library strategy miRNA-Seq
Library source transcriptomic
Library selection size fractionation
Instrument model Illumina HiSeq 2000
Description small RNA profiling
Data processing Illumina Casava 1.7 software used for basecalling
Sequencing data was filtered through a pipeline of UEA small RNA workbench 3.0 with default parameters (Stocks et al. 2012). The sequences without 3’ adaptor and those trimmed sequences not lying between the specified sequence lengths were discarded and sequences with the length specified by the user (16-35 nt) were extracted. Further, low complexity sequences (sequences containing less than 3 distinct nucleotides), invalid tags with ‘N’ nucleotides, degradation products and other non-small RNAs were discarded. The fragments of rRNA, tRNA, snRNA or snoRNA were removed after being mapped on plant t/rRNAs sequences from “Rfam” (except miRNA), Arabidopsis tRNAs from “The Genomic tRNA Database”, and plant t/rRNA sequences from “EMBL” release 95.
For identification of miRNAs and their precursors, the clean putative small RNA reads obtained after filtration through elimination pipeline were aligned to tomato reference genome dataset (Solanaceae Genomics Project Assembly SL2.40 obtained from NCBI) (The Tomato Genome Consortium) with no mismatch by using miRCat pipeline of UEA sRNA tool kit with set parameters (Stocks et al. 2012).
Genome_build: Solanaceae Genomics Project Assembly SL2.40
Supplementary_files_format_and_content: The .txt files report abundance measurements of small RNA reads
Submission date Oct 05, 2016
Last update date May 15, 2019
Contact name Gopal Joshi
Phone +91-11-27662609
Organization name Delhi University
Department Department of Botany
Lab Plant Genomics & Stress Biology
Street address First Floor
City New Delhi
State/province Delhi
ZIP/Postal code 110007
Country India
Platform ID GPL16345
Series (1)
GSE87651 Genome-wide identification and characterization of miRNAome from tomato roots (Solanum lycopersicum) and root-knot nematode (Meloidogyne incognita) during susceptible and resistant interactions.
BioSample SAMN05864229
SRA SRX2213273

Supplementary file Size Download File type/resource
GSM2337661_Stage1_Uninfected_processed.txt.gz 17.1 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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