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Sample GSM3190077

Query DataSets for GSM3190077

Status

Public on Jun 15, 2018

Title

L1-1

Sample type

SRA

Source name

Stem from jointing stage of low hybrid

Organism

Zea mays

Characteristics

tissue: Stem
developmental stage: jointing stage
hybrid/phenotype: low (L, Dan 598xFAPW)

Treatment protocol

Eighteen samples from the low, middle and high hybrid at three development stages were used for RNA sequencing.

Growth protocol

Three different maize hybrids, with extremely distinct plant height, which were further classified into low (L), middle (M) and high (H) group, were selected for RNA sequencing at three key developmental stages, namely, jointing stage (I), big flare period (II) and tasseling stage (III).

Extracted molecule

total RNA

Extraction protocol

1 μg of total RNA was subjected to library synthesis using the TruSeq V2 RNA-Seq kit. 1 μg of total RNA was enriched for the Poly-A mRNA and reverse transcribed to double-stranded cDNA. The cDNA was end repaired, adenylated, and ligated with Illumina indexes prior to 12 cycles of PCR. Sequencing was performed using 12pM hybridization to a 2x100 paired end flow cell.
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

Data processing

The raw sequencing reads from all the runs were first trimmed for lo- quality regions and adapter sequences. Reads containing N (indicating uncertain base information) greater than 10% were removed, and low quality reads were removed. Clean reads (~90% of the total read count) were mapped to the maize genome (B73_RefGen_v4 annotation build, http://plants.ensembl.org/Zea_mays/Info/Index) and a recent internal maize transcriptome using Tophat 2 with default parameters (Pertea 2010). The DESeq Bioconductor package (Version: 1.28.0) was used to normalize the mapped reads. Data quality analysis was evaluated by visualizing the results of principal component analysis (PCA).
The mapped sequence reads for all of the identified genes were used for differential expression analysis by the DESeq method. Using Cufflinks 2.2.1 and Cuffdiff, gene expression levels were calculated as reads per kilobase of transcript sequence per million base pairs sequenced (FPKM) (Trapnell et al. 2016). Based on statistical analyses, genes with a P-value < 0.05 and an absolute value |log2 ratio ≥ 1| were considered to be significant differentially expressed genes (DEGs). GO enrichment analysis was conducted using Blast2GO (https://www.blast2go.com/) with FDR < 0.05. Based on their expression patterns, the DEGs were clustered by the Genesis K-means method
Reads Per Kilobase of exon per Megabase of library size (RPKM) 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.

Submission date

Jun 14, 2018

Last update date

Jun 15, 2018

Contact name

Hengsheng Wang

E-mail(s)

[email protected]

Organization name

Anhui Agriculture University

Street address

Changjiang Rode 130