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Sample GSM1708738

Query DataSets for GSM1708738

Status

Public on Dec 30, 2021

Title

EN 0DAP rep1

Sample type

SRA

Source name

Kernel

Organism

Zea mays

Characteristics

strain: B73
tissue: Endosperm
developmental stage: 0 DAP

Treatment protocol

N/A

Growth protocol

Maize (Zea mays) inbred line B73 plants were grown in a greenhouse with a cycle of 16 h of light at ~30°C and 8 h of dark at ~25°C at the University of Arizona during June-October 2012. The unfertilized or self-pollinated ears were harvested 0 to 4 days after pollination for laser-capture microdissection of the RNA. The tissue sections for each biological replicate of each stage of endosperm development and the embryo (samples) were pooled from multiple kernels of a single ear.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated using the Arcturus PicoPure RNA Isolation kit (Applied Biosystems), treated with TURBO DNase (Ambion), and purified with the Arcturus PicoPure kit again. RNA reverse transcription with oligo(dT) and random primers, second-strand cDNA synthesis, purification, cDNA amplification and second-purification were using the Nugen ovation RNA-Seq kit v2 (Nugen), XP-beads (Beckman Coulter) and the MinElute PCR Purification kit (Qiagen) following the manufacturer’s instructions. In RNA purification of the central cell/early endosperm stages (0-2 DAP) and the embryo (4 DAP), we repeatedly found indications of a carryover product(s) that caused an overestimation of RNA measurements using standard procedures. Instead, we used the height of the 18S-rRNA band from the Bioanalyzer 2100 for measuring the RNA concentration. In all these cases, ~5μg of amplified cDNA was obtained from ~10 ng of captured RNA.
The cDNA paired-end libraries construction and quality test were prepared using TruSeq DNA Sample Preparation kit v2 (Illumina) with around 1μg amplified cDNA by following the manufacturer’s suggested procedures.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Illumina HiSeq 2500 System
Because of a failure of two samples within the first bio-replicate set, all samples for this bio-replicate were sequenced a second time from the library construction step through RNA-Seq, and the resulting total reads from both runs were combined for the subsequent analyses.
Raw reads were trimmed using the Trimmomatic program to remove adapter sequences and low-quality bases.
Trimmed and high quality reads were aligned to the maize B73 reference genome using TopHat v2.0.9. Intron length was set to 30 to 8000 nt with maximum number of mismatches per read set to 3 (-i 30 -I 8000 -N 3).
Raw count table of exonic reads built by BEDTools v2.17.0 with the exon coordinates (GTF, EnsemblPlants) were used in the software edgeR, reported as counts per million mapped reads (CPM), and filtered with a lower limit of ≥0.5 in at least three of the 18 samples.
The filtered raw count table was further normalized with the trimmed mean of M-values (TMM) method across the 18 samples, and transformed to CPM table using edgeR.
Genome_build: the maize B73 reference genome (AGPv3.20)
Supplementary_files_format_and_content: Tab-delimited text files include CPM values of gene for all 18 samples.

Submission date

Jun 11, 2015

Last update date

Dec 30, 2021

Contact name

Ramin Yadegari

E-mail(s)

yadegari@email.arizona.edu

Phone

520-621-1616

Organization name

University of Arizona