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Sample GSM4547739

Query DataSets for GSM4547739

Status

Public on Oct 28, 2020

Title

Leaves, RB-3 (Transcriptome)

Sample type

SRA

Source name

Leaves, RB

Organism

Oryza sativa

Characteristics

strain: NIP
tissue: leaves
infection: Rice black-streaked dwarf virus (RBSDV)
Stage: 30 days after inoculation

Treatment protocol

RBSDV were transmitted experimentally to 10-day-old seedlings by the small brown plant hopper (Laodelphax striatellus) for 3 days.

Growth protocol

Rice plants were then grown in a glasshouse with a condition of a 14/10 light/dark cycle under artificial light at 28-30 °C

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0. Approximately 10 ug of total RNA representing a specific adipose type was subjected to isolate Poly (A) mRNA with poly-T oligoattached magnetic beads (Invitrogen). Following purification, the poly(A)- or poly(A)+ RNA fractions is fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the protocol for the mRNA-Seq sample preparation kit (Illumina, San Diego, USA), the average insert size for the paired-end libraries was 300 bp (±50 bp). And then we performed the paired-end sequencing on an Illumina Hiseq 4000 at the (lc-bio, China) following the vendor's recommended protocol.
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

4_genes_fpkm_expression.txt

Data processing

Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to Osativa_323_v7.0 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
Reads Per Kilobase of exon per Megabase of library size (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.
Data filtering steps.Prior to assembly, the low quality reads(1,reads containing sequencing adaptors; 2,reads containing sequencing primer;3, nucleotide with q quality score lower than 20 )were removed .software CutAdapter,parameters -o 5 -p 100
Read alignment software,software HISAT ,version 2.0 ,parameters -l fr-firststrand -mi 20 -mx 500000 -p 2 -b dta -q phred33-quals -x 9
perform expression level for mRNAs by calculating FPKM.stimate the expression levels of all transcripts.software StringTie ,version 1.3.0,parameters -b dta -q phred33-quals.
Genome_build: Osativa_323_v7.0
Supplementary_files_format_and_content: tab-delimited text file includes FPKM expression values for each Sample

Submission date

May 12, 2020

Last update date

Oct 28, 2020

Contact name

Linying Li

E-mail(s)

lilinying6297@126.com

Organization name

Zhejiang Academy of Agricultural Sciences

Street address

198 Shiqiao Road