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Sample GSM4795296

Query DataSets for GSM4795296

Status

Public on Sep 18, 2023

Title

TaF24T

Sample type

SRA

Source name

inoculated with Fusarium graminearum for 24 hours

Organism

Triticum aestivum

Characteristics

cultivar: LiangXing66
tissue: coleoptile
inoculation status: inoculated with Fusarium graminearum
time: 24 hours

Treatment protocol

Coleoptiles were inoculated with 3 µL of conidia suspension (1.0 x 106 spores/mL) and then were returned into the incubator. After 12 and 24 hpi, the coleoptile inoculation sites about 3 mm were harvested. At the same time, coleoptiles treated with distilled water were collected as mock treatments. Once harvested, all samples were frozen in liquid nitrogen as quickly as possible and stored at -80 °C for RNA-Seq.

Growth protocol

T. aestivum seeds were grown in a light incubator at 25 °C for two and a half days, under a photoperiod of 16 h light and 8 h dark, then the tips of the coleoptile were cut off quickly with aseptic scissors for F. graminearum inoculation.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted using Trizol reagent.
A total amount of 20 ng RNA per sample was used as input material for the RNA sample preparations. Firstly, ribosomal RNA was removed by Epicentre Ribo-zero™ rRNA Removal Kit (Epicentre, USA), and rRNA free residue was cleaned up by ethanol precipitation. Subsequently, sequencing libraries were generated using the rRNA-depleted RNA by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. Briefly, 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(RNaseH-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. In the reaction buffer, dNTPs with dTTP were replaced by dUTP. 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 150~200 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, products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

LiangXing66,China, winter wheat

Data processing

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 on containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content of the clean data were calculated. All the down stream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using HISAT2 v2.0.4 and paired-end clean reads were aligned to the reference genome using HISAT2(Langmead, B.et al) v2.0.4. HISAT2 was run with ‘--rna-strandness RF’, other parameters were set as default.
The mapped reads of each sample were assembled by StringTie (v1.3.3) (Mihaela Pertea.et al. 2016) in a reference-based approach. StringTie uses a novel network flow algorithm as well as an optional de novo assembly step to assemble and quantitate full-length transcripts representing multiple splice variants for each gene locus.
Genome_build: IWGSC 1.0, FASTA provided for TUCP and lncRNA on series record.
Supplementary_files_format_and_content: fpkm expression values

Submission date

Sep 18, 2020

Last update date

Sep 18, 2023

Contact name

Xiaoxin Duan

E-mail(s)

2017202047@njau.edu.cn

Organization name

Nanjing Agricultural University

Street address

No.1 Weigang, Xuanwu District