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Status |
Public on Jan 01, 2021 |
Title |
Leaf Plenodomus tracheiphylus treated rep1 |
Sample type |
SRA |
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Source name |
Leaf
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Organism |
Citrus jambhiri |
Characteristics |
treatment: Inoculum containing 10^6 mL-1 phialoconidia of Plenodomus tracheiphilus PT10 strain tissue: leaf
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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
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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
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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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Description |
Pt_1
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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
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Submission date |
Dec 31, 2020 |
Last update date |
Jan 01, 2021 |
Contact name |
Angela Roberta Lo Piero |
E-mail(s) |
rlopiero@unict.it
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Phone |
+39-0957580238
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Organization name |
University of Catania
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Department |
Di3A
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Lab |
Plant genetics
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Street address |
Via S. Sofia 98
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City |
Catania |
State/province |
CT |
ZIP/Postal code |
95123 |
Country |
Italy |
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Platform ID |
GPL29550 |
Series (1) |
GSE164096 |
De novo transcriptome sequencing of rough lemon leaves (Citrus jambhiri Lush.) in response to Plenodomus tracheiphilus infection |
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Relations |
BioSample |
SAMN17190621 |
SRA |
SRX9763513 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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