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Sample GSM4996597

Query DataSets for GSM4996597

Status

Public on Jan 01, 2021

Title

Leaf control rep2

Sample type

SRA

Source name

Leaf

Organism

Citrus jambhiri

Characteristics

treatment: Water
tissue: leaf

Treatment protocol

The inoculum was prepared as follows: three pieces of young fungus grown at 21°C ± 2 in Petri dishes containing potato dextrose agar medium (PDA) were placed in 7 different flasks containing 100 mL of carrot broth and incubated for 5 days in a heidolph unimax 2010 shaker at 22 °C. Successively, the growth medium was filtered and centrifugated at 8.000 rpm x 20 min. The pellet was recovered and the phialoconidia were counted with a counting chamber to adjust the inoculum concentration at 106 mL-1. The inoculation of Pt plants was performed by depositing 10 µl on wounds obtained by cutting the midvein of three leaves for each plant with a sharp sterile blade. CK plants were inoculated with sterile water

Growth protocol

Seeds of rough lemon (C. jambhiri) were sowed on sterile peat in May 2019. After 6 months of growing in a chamber at 25 °C and 90% humidity, Pt plants were inoculated with the pathogen Plenodomus tracheiphilus PT10 strain

Extracted molecule

total RNA

Extraction protocol

Both inoculated and control samples were collected 15 days after inoculation. Leaves were immediately frozen with liquid nitrogen and stored at -80°C until both DNA and RNA extractions were performed. DNA extraction was performed according to Springer 2010. The RNA was extracted using the RNeasy® Plant Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions.
All samples passed through three steps before library construction: Nanodrop for preliminary quantitation, agarose gel electrophoresis to tests RNA degradation and potential contamination, Agilent 2100 to checks RNA integrity and quantitation. After the QC procedures, mRNA from is enriched from total RNA using oligo(dT) beads. The mRNA is then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) is added, with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation and AMPure XP beads is used to purify the cDNA. The final cDNA library is ready after a round of purification, terminal repair, Atailing, ligation of sequencing adapters, size selection and PCR enrichment. Libraries are fed into Illumina machines according standard Illumina protocol.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

CK2

Data processing

Base calling, base quality and Phred score relationship performed with the Illumina CASAVA v1.8 software
Raw reads are filtered to remove reads containing adapters or reads of low quality
Sequences putatively belonging to pathogen in inoculated rough lemon samples were removed by filtering out the reads mapped to the fungus genome (https://mycocosm.jgi.doe.gov/Photr1/Photr1.info.html).
In the absence of a reference genome clean reads have been assembled using Trinity software (K 25, Kmer coverage 2). Corset software was used for hierarchical clustering (parameter -m 10)
Seven databases have been applied for gene functional annotation: Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KEGG and GO.
CDS preduction using blast and EST-scan
De novo transcriptome filtered by Corset used as a reference for mapping reads back to transcriptome and quantify the expression level using RSEM.
DESeq used for gene expression difference analysis
Genome_build: FASTA of assembled transcripts is available on the series record.
Supplementary_files_format_and_content: Microsoft excell file containing raw readcounts of all unigenes in each sample

Submission date

Dec 31, 2020

Last update date

Jan 01, 2021

Contact name

Angela Roberta Lo Piero

E-mail(s)

rlopiero@unict.it

Phone

+39-0957580238

Organization name

University of Catania