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| Status |
Public on Dec 07, 2022 |
| Title |
C6, infected by S. sclerotiorum, 24hl, rep2 |
| Sample type |
SRA |
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| Source name |
leaf
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| Organism |
Helianthus annuus |
| Characteristics |
tissue: leaf
phenotype: DR
inbred line: C6
infection: infected by S. sclerotiorum, 24h
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from sunflower leaves using RNAprep Pure Plant Kit (TIANGEN Biotech, BeiJing, China) according to manufacturer’s instructions. RNA concentration and purity was measured using NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE).
A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer（5X）. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H . Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 240 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v4-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and paired-end reads were generated.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Helianthus annuus Linn.
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| Data processing |
The raw reads were further processed with a bioinformatic pipeline tool, BMKCloud(www.biocloud.net) online platform.
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data(clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30, GC-content and sequence duplication level of the clean data were calculated. All the downstream analyses were based on clean data with high quality.
The adaptor sequences and low-quality sequence reads were removed from the data sets. Raw sequences were transformed into clean reads after data processing. These clean reads were then mapped to the reference genome sequence. Only reads with a perfect match or one mismatch were further analyzed and annotated based on the reference genome. Hisat2 tools soft were used to map with reference genome.
Quantification of gene expression levelsGene expression levels were estimated by fragments per kilobase of transcript per million fragments mapped. Differential expression analysis of two conditions/groups was performed using the DESeq2. DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value < 0.01 found by DESeq2 were assigned as differentially expressed.
Assembly: Helianthus_annuus.HanXRQr1.0.genome.fa (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/002/127/325/GCF_002127325.1_HanXRQr1.0/GCF_002127325.1_HanXRQr1.0_genomic.fna.gz)
Supplementary files format and content: text files include FPKM and count values for each Sample
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| Submission date |
Dec 05, 2022 |
| Last update date |
Dec 09, 2022 |
| Contact name |
Mingzhu Zhao |
| E-mail(s) |
leesy2022@126.com
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| Organization name |
Liaoning Academy of Agricultural Sciences
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| Department |
Institute of Crop Research
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| Street address |
Dongling Roat
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| City |
Shenyang |
| State/province |
Liaoning |
| ZIP/Postal code |
118100 |
| Country |
China |
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| Platform ID |
GPL24801 |
| Series (1) |
| GSE220161 |
Comparative transcriptome analysis reveals the Sclerotinia sclerotiorum resistance mechanism in sunflower |
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| Relations |
| BioSample |
SAMN32063316 |
| SRA |
SRX18504855 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6792968_C6-infected_2.txt.gz |
321.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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