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Sample GSM6921285

Query DataSets for GSM6921285

Status

Public on Jan 08, 2023

Title

Baipimai, 48hour, rep1

Sample type

SRA

Source name

spike

Organism

Triticum aestivum

Characteristics

cultivar: Baipimai
tissue: spike
time: 48 hour

Extracted molecule

total RNA

Extraction protocol

The plant total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) according the instructions provided by the manufacturer.
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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

43-48-1

Data processing

Data process analysis was performed using BMKCloud (www.biocloud.net). Quantification of gene expression levelsGene expression levels were estimated by fragments per kilobase of transcript per million fragments mapped. The formula is shown as follow: FPKM=cDNAFragments/(MappedFragments(Millions)∗TranscriptLength(kb))
Assembly: NCBI
Supplementary files format and content: txt

Submission date

Jan 06, 2023

Last update date

Jan 08, 2023

Contact name

Cheng Kou

E-mail(s)

koucheng7417@outlook.com

Phone

+8615290564435

Organization name

Northwest A&F University