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Sample GSM1970278 Query DataSets for GSM1970278
Status Public on May 20, 2016
Title Pusa Ruby Infected 2
Sample type SRA
Source name Leaf tissue
Organism Solanum lycopersicum
Characteristics tissue: leaf
cultivar: Pusa Ruby
infection status: Alternaria infected
tissue appearance: Chlorotic/slightly blight
Treatment protocol Mock inoculation was done with the buffer (MES) in which Alternaria spore forming units were suspended. Plants were infected with a solution (~1000000 CFU/ml) of Alternaria using droplet inoculation method (~10 microlitre drop on leaves).
Growth protocol Tomato plants were grown under controlled environment, at 25ºC temperature. Prior to the infection plants were kept in a humid chamber for 24 hours. Alternaria fungus was grown on Potato Dextrose Agar plate, collected by scraping, homogenized, suspended in MES buffer and colony forming units (CFU) were counted.
Extracted molecule total RNA
Extraction protocol Three Trizol (Invitrogen) extracted RNA for the samples “Control 1, Control 2 and Infected 1” were used. For one sample “Infected 2”Agilent Plant RNA Isolation kit (#5188-2780) was used for RNA extraction. RNA purity was tested using Nanodrop 1000(Agilent Technologies).Integrity of RNA was assessed using Bioanalyzer 2100 (Agilent Technologies) on a RNA 6000 Nanochip. The QC criteria to pass the RNA quality on Bioanalyzer is RIN>7.The samples were found to be suitable for small RNA sequencing. For infected samples, only the infection zone tissues were collected.
Small RNA libraries for sequencing were constructed according to the IlluminaTruSeq Small RNA library protocol outlined in “TruSeq Small RNA Sample Preparation Guide” (Part # 15004197; Rev. A; Nov 2010). 1ug (microgram) of total RNA was used as the starting material. Briefly, 3’ adaptors were ligated to the specific 3’OH group of small RNA followed by 5’ adaptor ligation. The ligated products were reverse transcribed with Superscript II Reverse transcriptase by priming with Reverse transcriptase primers. The cDNA was enriched by 11cycles of PCR and cleaned using Polyacrylamide gel. The library was size selected in the range of 140 – 190 bp followed by overnight gel elution and salt precipitation using glycogen, 3M Sodium Acetate and absolute ethanol. The precipitate was re-suspended in resuspension buffer. The prepared library was quantified using Nanodrop and validated for quality by running an aliquot on High Sensitivity Bioanalyzer Chip (Agilent).The spread observed indicated an insert size of 22-70bp which is representative of smRNA fraction. Based on this observed Bioanalyzer profile they were decided to be suitable for sequencing.
Library strategy miRNA-Seq
Library source transcriptomic
Library selection size fractionation
Instrument model Illumina HiSeq 2000
Description Samples collected about 72 hours post infection
small RNA
Data processing Cutadapt was used to trim the adapter sequence from the fastq read sequences. sickle-master was used to pick reads above quality score of 30 and minimum read length of 18 bases. Reads of length greater than 25 nt were discarded.
The resulting reads were used to BLAST against known miRNAs from tfgd and other known plant miRNAs from mirbase. Reads with greater than 98% identity and alignment length greater than 18 were taken for further processing
Only the read alignment against the miRNA from the plant closest to sly was considered, for those reads aligning to multiple plant miRNAs other than sly. miRNA count was considered based on the number of reads aligning with the miRNA.
The process was repeated for all the samples.
DEseq was performed on the read counts, to find the differentially expressed miRNAs between the infected and control samples.
Genome_build: tfgd and mirbase
Supplementary_files_format_and_content: .txt files, representing the small RNA sequences with their corresponding read counts
Submission date Dec 11, 2015
Last update date May 15, 2019
Contact name Pallob Kundu
Phone 91-33-25693298
Organization name Bose Institute
Department Division of Plant Biology
Street address P 1/12 CIT Scheme VIIM
City Kolkata
State/province West Bengal
ZIP/Postal code 700054
Country India
Platform ID GPL16345
Series (1)
GSE75922 Genome-wide analysis of Alternaria stress-responsive microRNAs in tomato
BioSample SAMN04331789
SRA SRX1478067

Supplementary file Size Download File type/resource
GSM1970278_pusa_ruby_infected2_miRNA.txt.gz 1.2 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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