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Sample GSM1865355

Query DataSets for GSM1865355

Status

Public on Feb 09, 2017

Title

Zhewan1-25

Sample type

SRA

Source name

seed

Organism

Pisum sativum

Characteristics

cultivar: Zhewan 1
seed type: vegetable pea
harvest stage: 25 days after pollination

Growth protocol

The vegetable pea cultivar ‘Zhewan 1’ and the grain pea cultivar ‘Zhongwan 6’ were grown in the field of the Vegetable Research Institute, Zhejiang Academy of Agricultural Sciences. In Figure 1, the different developmental stages of the seeds of these two pea cultivars are shown. The pea pods were collected at different developmental stages (10, 15, 20, 25, and 30 days after pollination [DAP]). To control for the effect of biological variation, the materials that were collected for each sample were composed of large amounts from many individuals. The seeds from different plants were pooled together as one biological sample and three biological replicates were collected for each sample. The samples were frozen in liquid nitrogen immediately and stored at -80oC until further use.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using the TRIzol Reagent. The mRNA was isolated using oligo (dT)-attached magnetic beads. The four libraries were sequenced using IlluminaHiSeqTM 2000.
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

WD1

Data processing

The raw reads were first filtered by removing the adapter sequences and the low quality sequences, which included the reads with the N percentage (i.e., the percentage of nucleotides in a read that could not be sequenced) that was over 5% and the ones that contained more than 20% of the nucleotides in the read with a Q-value ≤ 10. The Q-value represented the sequencing quality of the related nucleotides. The clean reads were used in the de novo assembly of the transcriptomes. The RNA-Seq data were de novo assembled using the Trinity assembly program (Grabherr et al., 2011).
The function of the unigenes was annotated by BLASTing with an E-value threshold of 10-5 to protein databases that included the NCBI non-redundant (nr) database, the Swiss-Prot protein database, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and the Clusters of Orthologous Groups of proteins (COG) database. The proteins with the highest sequence similarity were retrieved for the analyses. The KEGG produced an annotation of the metabolic pathways, while the COG matched each annotated sequence to an ancient conserved domain, which represented the primary phylogenetic lineages of the pea. Based on the nr annotation, 6 top-hit species were identified, and the Gene Ontology (GO) classifications were obtained with Blast2GO.
The alignment software, bowtie 0.12.8, was used to map the reads back to the transcriptome. Then, the number of mapped clean reads for each unigene was counted and normalized into a RPKM value (Reads Per kb per Million reads), which is widely used to calculate the unigene expression (Mortazavi et al., 2008). To detect differentially expressed genes, statistical analyses among libraries were performed following the formula as described (Shen et al., 2012), where FDR (false discovery rate) were used to determine the threshold the P value in multiple tests and analyses using the q-value package (Benjamini et al., 2001). In our work, the differentially expressed unigenes between two samples were screened with the threshold of FDR ≤ 0.001 and the absolute value of log2Ratio ≥ 1. Furthermore, the GO classifications and the KEGG pathway enrichments were compared between up-regulated and down-regulated unigenes.
Supplementary_files_format_and_content: [Expression_data_differential_analysis.xlsx] expression data of genes and differential analysis results, and function of interest of some unique genes
Supplementary_files_format_and_content: [Transcript_expression_total.txt] expression data for all Unigenes
Supplementary_files_format_and_content: Trinity_uniq.fasta is linked as a supplementary file on the Series record.

Submission date

Sep 01, 2015

Last update date

May 15, 2019

Contact name

Na Liu

E-mail(s)

[email protected]

Phone

8657186416055

Organization name

Zhejiang Academy of Agricultural Sciences