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Sample GSM3502437

Query DataSets for GSM3502437

Status

Public on Dec 31, 2019

Title

PS_Control

Sample type

SRA

Source name

Aerial tissues

Organism

Phaseolus vulgaris

Characteristics

genotype: wild type
cultivar: Pinto Saltillo
condition: Control
description: FRI-040-251104
age: Forty-five-day-old seedlings

Treatment protocol

Forty-five-day-old seedlings showing three trifoliate leaves were assigned into three groups for the experiment, namely plants without any treatment (Control), drought-treated plants (Drought) and post-drought recovery plants (Recovery). Whereas plants of the control group (C) grew under continuous irrigation, drought-treated plants (D) were submitted to a period of progressive water deﬁcit for two weeks by suppression of irrigation. Some plants subjected to the drought treatment were re-watered with Hoagland´s solution to allow plant recovery and classified as post-drought recovery plants (R).

Growth protocol

Seeds were washed five times with sterile distilled water before planting in sterile aluminum trays containing a layer of wet sheets of paper. Trays were covered with aluminum foil and incubated at 30°C for a week. Then, seedlings were transferred into plant pots and incubated in greenhouse conditions using sterile vermiculite as substrate. All plants were watered with Hoagland´s basal salt solution in increasing concentrations every week (from 0.1X to 1X).

Extracted molecule

total RNA

Extraction protocol

Pooled plant samples were powdered by grinding the frozen tissue. Each pool included 6-9 plants of each cultivar under control conditions or drought treatment. Thus, 12 RNA samples were extracted in two replicates under either drought or control conditions. Total RNA was extracted using 200 mL of the powdered sample and adding 700 mL of the Z6-extraction buffer (8 M guanidinium-HCl, 20 mM MES, 20 mM EDTA, 50 mM β-mercaptoethanol, pH 7.5). Then, an equal volume of phenol was added to carry out the extraction of RNA, followed by purification using the ssDNA/RNA Clean & Concentrator™ kit (Zymo Research Corp, Orange, CA, USA), according to the manufacturer's instructions. Equal amount of RNA from control and drought conditions were pooled together for further analysis, resulting in two RNA populations. The integrity of RNA was checked by agarose gel electrophoresis and the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA). The concentration of total RNA was determined using the NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE).
Two libraries were constructed for each treatment using the TruSeq RNA Sample Preparation Kit (Illumina, Inc., San Diego, US-CA), following the manufacturer’s recommendations. In brief, poly(A)-tailed mRNA was enriched and fragmented, followed by first cDNA synthesis. Subsequent second strand cDNA synthesis and the final reactions were cleaned up prior to perform the end repair step, and the addition of a single adenylate into the 3´ends. Adapters were ligated to both ends of the short fragments, which were enriched by 36 PCR cycles prior to be cleaned up and validated. cDNA fragments pools were loaded to Illumina MiSeq (Illumina, Inc., San Diego, US-CA) platform for single-ended sequencing.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Data processing

Illumina CASAVA-1.8.2 software used for basecalling.
Sequenced reads were trimmed for adaptors and then data were quality-assessed using the FastQC tool. Low-quality reads were discarded, and the generated clean data were aligned to the reference genome of P. vulgaris (G19833) (Schmutz et al. 2014) using TopHat (Trapnell et al. 2009).
Cuffdiff was used to compare the expression level of transcripts, and to test the statistical significance between two conditions (Trapnell et al. 2012). To identify differentially expressed genes (DEGs), genes were ranked according to normalized fragments per kilobase per million mapped reads (FPKM). FPKM values were assigned to each gene by comparing the FPKM value under the drought treatment to that in the control condition. Genes that were induced or reduced at least 2-fold change (log2) and P value ≤ 0.05 were identified as DEGs by Cuffdiff (Ghosh and Chan 2016).
Genome_build: P. vulgaris (G19833)
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each sample …

Submission date

Dec 06, 2018

Last update date

Dec 31, 2019

Contact name

Josefat Gregorio Jorge

E-mail(s)

josefatgregorioj@gmail.com

Organization name

Instituto Politécnico Nacional

Department

Biotecnología Genómica