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Sample GSM2133750

Query DataSets for GSM2133750

Status

Public on Apr 27, 2016

Title

IS20351_WW_1

Sample type

SRA

Source name

Leaf of IS20351 well-watered condition

Organism

Sorghum bicolor

Characteristics

tissue: Leaf
genotype: IS20351
irrigation status: well-watered

Treatment protocol

Forty plants were divided in two groups: the well-watered (WW) and the drought stressed (DS) plants. Irrigation was withheld for half of them (the DS ones) till the FTSW value reached 0.2. This value was kept constant for nine days by re-integrating water losses of the DS plants day by day, while the WW plants were irrigated daily to maintain soil water content close to 0.7 FTSW. After nine days had passed, the plants were harvested in order to perform physiological and transcriptomic analysis

Growth protocol

Germination of seeds was carried out in Petri dishes at 25 °C and in dark conditions for 3 days. Five germinated seeds were planted in plastic pots (16 L capacity), filled with a base layer of sand to guarantee drainage and 8 kg of a soil mixture (24% clay, 64% silt, and 12% sand), that had been previously sieved, dried and homogenized. At the 4th leaf stage, plants were thinned in order to have one healthy plant per pot. Plants were grown under well-water conditions until they reached the 6th leaf stage. At this moment, all the plants were irrigated until FC, the soil surface was covered by a thin layer of perlite, and the top of the pot was covered with PVC bags. A little slit was made in the bottom of the plastic bag to allow the sorghum plant to grow through. The slit was sealed with adhesive packing tape to minimise water loss by evaporation. Following the protocol of the dry-down experiment (Fracasso et al., 2016) a decrease of pot weight between two consecutive weight determinations is only attributed to plant transpiration.

Extracted molecule

total RNA

Extraction protocol

Three biological replicates were used for all RNA-Seq experiments from each genotypes and water treatment. The total RNA from the leaf meristem was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) and purified using the RNeasy Plant Mini kit (Qiagen, Valencia, CA). On column DNase digestion was performed according to the manufacturer’s protocol (Qiagen, Valencia, CA). RNA quality and integrity was verified using a 2100 Bioanalyzer RNA Nanochip (Agilent, Santa Clara, CA) and all three samples had RNA Integrity Number (RIN) value more than 8.5. The quantification of the total RNA was checked by a NanoDropND-1000 Spectrophotometer (Nano-Drop, Wilmington, DE) and agarose gel electrophoresis.
Illumina sequencing using the GAII platform was performed at Beijing Genomics Institute (BGI-Shenzhen, Shenzhen, China http://www.genomics.cn/index.php) according to the manufacturer’s instructions (Illumina, San Diego, CA). Briefly, poly-A RNA was isolated from 20 μg of total RNA using Magnetic Oligo (dT) Beads (Illumina) and digested in short fragment. First and second strand synthesis were followed by end repair, and adenosines were added to the 3’ ends. Adapters were ligated to the cDNA and fragments (200 ± 25 bp) were purified by agarose gel electrophoresis and amplified by PCR. Finally, after validating on an Agilent Technologies 2100 Bioanalyzer using the Agilent DNA 1000 chip kit, the cDNA library was sequenced on a PE flow cell using Illumina Genome AnalyzerIIx, and the workflow was as follows: template hybridization, isothermal amplification, linearization, blocking, sequencing primer hybridization, and sequencing on the sequencer for Read 1

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer IIx

Description

processed data file: Genotype1-control.Gene.rpkm.xls

Data processing

OLB-1.9.4 software was used for basecalling
Raw reads were filtered to remove the sequence of adapter, content of unknown bases higher than 10% per reads, and low quality reads. After filtering, the remaining reads are called "clean reads" and used for downstream bioinformatics analysis.
Clean reads are mapped to a reference sequences using SOAPaligner/SOAP2. No more than 5 mismatches are allowed in the alignment
Gene expression levels were calculated by using the RPKM method (Reads per kilobase transcriptome per million mapped reads) according to Mortazavi, A., B. A. Williams, et al. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods. 2008.5(7): 621-8.
Genome_build: ftp://ftp.ensemblgenomes.org/pub/plants/release-20/fasta/sorghum_bicolor/dna/
Supplementary_files_format_and_content: Genotype1-control.Gene.rpkm.xls: the excel file include the number of unique mappede reads, lenght and RPKM for each gene found in the genotype IS20351 under well-watered conditions.
Supplementary_files_format_and_content: Genotype1-treatment.Gene.rpkm.xls: the excel file include the number of unique mappede reads, lenght and RPKM for each gene found in the genotype IS20351 under drought-stressed conditions
Supplementary_files_format_and_content: Genotype2-control.Gene.rpkm.xls: the excel file include the number of unique mappede reads, lenght and RPKM for each gene found in the genotype IS22330 under well-watered conditions
Supplementary_files_format_and_content: Genotype2-treatment.Gene.rpkm.xls: the excel file include the number of unique mappede reads, lenght and RPKM for each gene found in the genotype IS22330 under drought-stressed conditions

Submission date

Apr 26, 2016

Last update date

May 15, 2019

Contact name

Alessandra Fracasso

E-mail(s)

alessandra.fracasso@unicatt.it

Organization name

Università Cattolica del Sacro Cuore

Department

DiProVeS