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Sample GSM2904733

Query DataSets for GSM2904733

Status

Public on Feb 15, 2018

Title

Col-0-1 rep1

Sample type

SRA

Source name

Developing seeds

Organism

Arabidopsis thaliana

Characteristics

tissue: Developing seeds
development stage: embryo development
genotype: wild type

Growth protocol

Arabidopsi grown in full-spectrum white fluorescent light at 22°C under long day conditions (16 h light/8 h dark).

Extracted molecule

total RNA

Extraction protocol

Developing seeds at 9 DAP were dissected from siliques by needle, and total RNA was extracted by Plant RNA Kit (R6827, Omega).
A total amount of 3 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer（5X） . First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase（RNase H-） . Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Data processing

Illumina Casava1.8 software used for basecalling.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Bowtie v2.2.3 and paired-end clean reads were aligned to the reference genome using TopHat v2.0.12.
HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.
Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq R package (1.18.0). The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate . Genes with an adjusted P-value <0.05 found by DESeq were assigned as differentially expressed.
GO analysis was performed by the GO Annotation of TAIR.
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample

Submission date

Dec 26, 2017

Last update date

Feb 15, 2018

Contact name

yilong hu

E-mail(s)

[email protected]

Organization name

south china botanical gardern, chinese academy of science

Street address

No.723, xingke road, tianhe district