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Sample GSM5840796

Query DataSets for GSM5840796

Status

Public on Feb 03, 2022

Title

Root control replicate 2

Sample type

SRA

Source name

Root

Organism

Arundo donax

Characteristics

treatment: Water + 5.774 grams/L of ammonium nitrate

Treatment protocol

The contamination of each pots was achieved by using 4 ppm (4 mg/kg) of cadmium nitrate Cd(NO3)2 for treated plants and 5.774 grams/L of NH4NO3 for control plants, so as to equilibrate the N concentration between the two conditions.

Growth protocol

The trial started on May 4th 2020, by filling each pot with 8 kg of clay soil and kept in open air. Afterwards, one litre of tap water was added to each pot to allow the element adsorption by soil colloids. Successively, in each pot a single A. donax stem node was transplanted and the irrigation was performed three times a week by adding one litre of tap water. The pots were arranged according to a randomized block factor scheme, considering three biological replicates for each treatment, with a total of six experimental units.

Extracted molecule

total RNA

Extraction protocol

On July 28th both treated and control samples were collected after prolonged cadmium exposure. Leaves and roots were immediately frozen with liquid nitrogen and stored at -80°C until further uses. RNA isolation was carried out by using the Spectrum Plant Total RNA Extraction kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions.
All samples passed through three steps before library construction: Nanodrop for preliminary quantitation, agarose gel electrophoresis to tests RNA degradation and potential contamination, Agilent 2100 to checks RNA integrity and quantitation. After the QC procedures, mRNA from is enriched from total RNA using oligo(dT) beads. The mRNA is then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) is added, with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation and AMPure XP beads is used to purify the cDNA. The final cDNA library is ready after a round of purification, terminal repair, Atailing, ligation of sequencing adapters, size selection and PCR enrichment. Libraries are fed into Illumina machines according standard Illumina protocol.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Base calling, base quality and Phred score relationship performed with the Illumina CASAVA v1.8 software
Raw reads are filtered to remove reads containing adapters or reads of low quality
In the absence of a reference genome clean reads have been assembled using Trinity software (minKmerCov= 3, min_glue=4). Corset software was used for hierarchical clustering (parameter -m 10)
Seven databases have been applied for gene functional annotation: Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KEGG and GO.
CDS preduction using blast and EST-scan
De novo transcriptome filtered by Corset used as a reference for mapping reads back to transcriptome and quantify the expression level using RSEM.
DESeq2 used for gene expression difference analysis
Genome_build: FASTA assembled sequences
Supplementary_files_format_and_content: Microsoft excell file containing raw readcounts of all unigenes in each sample

Submission date

Jan 27, 2022

Last update date

Feb 03, 2022

Contact name

Angela Roberta Lo Piero

E-mail(s)

rlopiero@unict.it

Phone

+39-0957580238

Organization name

University of Catania