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Sample GSM2532796

Query DataSets for GSM2532796

Status

Public on Jun 29, 2017

Title

Yellow-Leaf 1

Sample type

SRA

Source name

plants leaves

Organism

Oryza sativa

Characteristics

genotype/variation: yl-1 mutant
background: EMS-induced mutant library of indica super early-season rice cv. Zhongjiazao 17
tissue: plants leaves

Treatment protocol

The seedlings were in a growth chamber (12/12h light/dark; light intensity 300 μmol m−2 s−1) at a constant temperature of either 26 ºC

Growth protocol

The seedlings were cultivated in 1/2 MS medium

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure
Approximately 5 ug of total RNA representing a specific adipose type was subjected to isolate Poly (A) mRNA with poly-T oligo attached magnetic beads (Invitrogen). Following purification, the mRNA 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).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

mRNA sequencing

Data processing

Prior to assembly, we removed the low quality reads(1,reads containing sequencing adaptors; 2,reads containing sequencing primer;3, nucleotide with q quality score lower than 20) in the raw data. And then we aligned reads of sample yl-1 and sample wild-type to the JGI (https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Osativa) rice reference genome using Tophat package, which initially remove a portion of the reads based on quality information accompanying each read and then maps the reads to the reference genome. Tophat allows multiple
alignments pe read (up to 20 by default) and a maximum of two mismatchs when mapping the reads to the reference.Tophat build a database of potential splice junctions and confirms these by comparing the previously unmapped reads against the database of putative junctions.
Transcript abundance estimation and differentially expressed testing
The aligned read files were processed by Cufflinks, which uses the normalized RNA-seq fragement counts to measure the relative abundances of the tianscripts. The unit of measurement is Fragment Per Kilobase of exon per Million fragments mapped (FPKM).The feference GTF annotation file used in Cufflinks was downloaded from the JGI database. Cufflink was used to de novo assemble the transcripiome at first; The second ,Cuffmerge was used to comerge all transcripts of sample A and B to generate unique transcripts. The downloaded JGI GTF file was passed to Cuffdiff along with the original alinment (SAM) files produced by Tophat. Cuffdiff re-estimates the abundance of the transcripts listed in the GTF file using aligments from the SAM file and concurrently test for different expression. Only the comparisons with “q_value”less than 0.05 and status marked as “OK” in the Cuffdiff output were regarded as showing differential expression.
Genome_build: Oryza sativa v7_JGI (Rice)
Supplementary_files_format_and_content: excel,expression profiles

Submission date

Mar 10, 2017

Last update date

May 15, 2019

Contact name

lv yu song

Organization name

CNRRI

Street address

CNRRI

City

Hangzhou