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| Status |
Public on Jun 10, 2021 |
| Title |
Rice Near Isogenic Line GRH |
| Sample type |
SRA |
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| Source name |
Leaf
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| Organism |
Oryza sativa |
| Characteristics |
line: Introgression
genotype: Near Isogenic line
tissue: Leaf
age: 21 days after germination
treatment: Green rice leafhopper (GRH)
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| Treatment protocol |
Seedlings with a uniform height were placed in test tubes after washing the loose soil from the roots. Seedlings were infested with second instar nymphs green rice leafhoppers for 48 h at room temperature as described earlier (Park et al., 2013). Leaf samples for RNA-Seq analysis were collected and sent for sequencing (Macrogen, Daejeon, Korea).
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| Growth protocol |
Two rice lines, Ilpum (the GRH susceptible cultivar) and a near isogenic line (NIL) carrying the Grh1 (a gene conferring resistance towards GRH) derived from a cross between Shingwang (indica, P1) and Ilpum (japonica, P2) (Park et al., 2013) were use as genetic materials to perform the experiments. Prior to germination, rice seeds were surfaced sterilized using nitric acid (HNO3, 0.7%) (CAS: 7697-37-2, Junsei Chemical Co. Ltd, Tokyo, Japan) overnight to break the dormancy (Kabange et al., 2021), followed by incubation for 48 h at 27 °C to induced germination. Then, germinated rice seeds were grown in 50-well trays containing an enriched soil (company name) until 3 leaf-stage.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from leaf samples, and DNA contamination were eliminated using DNase. mRNA purification kit was used to prepare the library. RNA fragments were randomly purified for short read sequencing, followed by reverse transcription of fragmented RNA into cDNA. The adapters were ligated onto both ends of the cDNA fragments. After amplifying fragments using PCR, fragments with insert sizes between 200–400 bp were selected, and both ends of the cDNA were sequenced by the read length (paired-end sequencing).
RNA libraries were prepared for sequencing using standard Illumina protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
NO_1
Oryza sativa L.
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| Data processing |
Illumina HCS 3.3.52 software used for basecalling.
The total number of bases, reads, GC (%), Q20 (%), Q30 (%) were calculated for 4 samples. Trimmomatic program (Bolger et al., 2014) was used to remove adapter sequences and bases with base quality lower than three from the ends. Also using sliding window method, bases of reads that does not qualify for window size 4, and mean quality 15 are trimmed. Afterwards, reads with length shorter than 36bp are dropped to produce trimmed data.
For assembled genes, longest contigs of the assembled contigs were filtered and clustered into the non-redundant transcripts using CD-HIT-EST (Li et al., 2006; Li et al., 2001) . We defined these transcripts as ‘unigenes’ which are used for predicting the ORFs (Open Reading Frames), annotating against several known sequence databases, and analyzing differentially expressed genes (DEGs). ORF prediction for unigenes was performed using TransDecoder program to identify candidate coding regions within transcript sequence. After extracting ORFs that are at least 100 amino acids long, TransDecoder predicted the likely coding regions.
Fragments Per Kilobase of transcript per Million mapped reads (FPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
For functional annotation of the unigenes, the Gene Ontology (GO) database was applied to classify the annotated unigenes using BLASTX of DIAMOND with an E-value cut-off of 1.0E-5. Classification of GO terms was subsequently performed using in-house script. The GO terms belonging to biological process (BP), cellular component (CC) and molecular function (MF) were listed (Figure S2). The Oryza sativa japonica (Japanese rice) (RAPDB) was used for alignment.
To identify the proteins distributed in eukaryotic orthologous groups (KOG), clusters of orthologous groups (COGs), and non-supervised orthologous groups (NOGs), we carried out BLASTX of DIAMOND with an E-value cut-off of 1.0E-5 on EggNOG (Evolutionary genealogy of genes) database. The annotated unigenes were mapped to the annotation of the corresponding orthologous groups in EggNOG database.
For functional annotation of the unigenes, we carried out BLASTX of DIAMOND with an E-value cut-off of 1.0E-5 on Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Bi-directional best hit (BBH), which is a widely used method to infer orthology was used to search against the KEGG database to obtain the KO (reference pathway) number of the KEGG annotation. The KO number of the transcriptome was also obtained according to KEGG annotation. For functional annotation of the unigenes, we carried out BLASTX of DIAMOND with an E-value cut-off of 1.0E-5 on UniProt (Universal Protein Resource) database. In addition to capturing the core data mandatory for each UniProtKB entry (mainly, the amino acid sequence, protein name or description, taxonomic data and citation information), as much annotation information as possible were added. For functional annotation of the unigenes, we carried out BLASTX of DIAMOND with an E-value cut-off of 1.0E-5 on Pfam database. The Pfam database is a large collection of protein families, each represented by multiple sequence alignments and hidden Markov models (HMMs). Proteins are generally composed of one or more functional regions, commonly termed domains. The identification of domains that occur within proteins can therefore provide insights into their function.
Genome_build: Oryza sativa japonica (Japanese rice) (RAPDB)
Supplementary_files_format_and_content: *.gene_abund.anno.tab: Tab-delimited text files include FPKM and TPM values for each Sample.
Supplementary_files_format_and_content: *.gtf
Supplementary_files_format_and_content: Expression_Profile.IRGSP-1.0_transcript_exon_2018-11-26.gene.txt
Supplementary_files_format_and_content: Expression_Profile.IRGSP-1.0_transcript_exon_2018-11-26.transcript.txt
Supplementary_files_format_and_content: MapMan_NIL.xls
Supplementary_files_format_and_content: MapMan_Ilpum.xls
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| Submission date |
Jun 09, 2021 |
| Last update date |
Jun 10, 2021 |
| Contact name |
ROLLY NKULU KABANGE |
| E-mail(s) |
rollykabange@korea.kr
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| Phone |
01054134881
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| Organization name |
Rural Development Administration
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| Department |
Department of Southern Area Crop Science
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| Lab |
Paddy Crop Division
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| Street address |
Jompiljaero 20, Naei-dong
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| City |
Miryang |
| State/province |
Gyeongsangnamdo |
| ZIP/Postal code |
50424 |
| Country |
South Korea |
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| Platform ID |
GPL23013 |
| Series (1) |
| GSE176497 |
RNA-Seq and Electrical Penetration Graph Revealed the Role of Grh1-Mediated Activation of Defense Mechanisms towards Green Rice Leafhopper (Nephotettix cincticeps Uhler) Resistance in Rice (Oryza sativa L.) |
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| Relations |
| BioSample |
SAMN19644772 |
| SRA |
SRX11101960 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5366640_NO.gene_abund.anno.tab.gz |
715.1 Kb |
(ftp)(http) |
TAB |
| GSM5366640_NO.gtf.gz |
3.5 Mb |
(ftp)(http) |
GTF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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