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Sample GSM1566887

Query DataSets for GSM1566887

Status

Public on Aug 28, 2015

Title

B. juncea control (BC) (PAIRED-END)

Sample type

SRA

Source name

7-day old whole seedling

Organism

Brassica juncea

Characteristics

strain: var varuna
tissue: whole seedling
age: 7-day

Treatment protocol

Seedlings were grown for seven days and then subjected to various abiotic stresses. Drought stress was imposed for 3 h and 12 h by replacing water with high osmolality solution (300 mM mannitol). For imposing high temperature stress, seedlings were placed in a BOD incubator (Scientific systems, India) at 42°C for 30 min and 4 h. Entire seedlings (including the roots) were harvested after specified time intervals, snap frozen in liquid nitrogen and stored at -80°C. Untreated seedlings were taken as control.

Growth protocol

Seeds were surface sterilized with 2% sodium hypochlorite solution for 10 minutes (min) on a shaker and then washed five times with double distilled water for three min each. Sterile seeds were hydroponically grown on a muslin cloth wrapped around a small container in a growth chamber at 24°C±1 with 16 hours (h) day/8 h night photoperiod.

Extracted molecule

polyA RNA

Extraction protocol

Total RNA was isolated using GITC-based method from abiotic stress treated and untreated whole seedlings, independently for each time point. Extracted RNA was quantified using spectrophotometer and an aliquot of heat denatured RNA was electrophoresed on denaturing agarose gel to check its integrity. RNA extracted from two different time points were pooled in equimolar amounts and three RNA-Seq libraries- BC (control seedlings), BDS (drought stressed seedlings) and BHS (high temperature stressed) were prepared utilizing NEBNext RNA-Seq library preparation Master Mix Set for Illumina procured from NEB, USA.
Poly A+ RNA was isolated from 10 µg of total RNA using Sera-Mag beads (GE Healthcare, UK) and fragmented chemically at high temperature. Fragmented RNA was qualitatively and quantitatively checked on Bioanalyzer (Agilent, USA). 250 ng of fragmented RNA was used for first strand reverse transcription using random primers followed by second strand synthesis. The ends of double stranded cDNA were repaired and mono-adenylated. Paired end adapters were ligated using Rapid T4 DNA ligase and then size fractionated. Approximately, 350 bp size region was eluted and PCR amplified for 12 cycles. The quality and quantity of prepared libraries was evaluated utilizing Bioanalyzer (Agilent, USA) Ultra-deep parallel sequencing was performed using Illumina Genome Analyzer IIx at University of Delhi South Campus, Delhi, India according to manufacturer’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer IIx

Data processing

Illumina Casava1.7 software used for basecalling.
RNA-Seq raw reads were processed by NGS-QC toolkit and low-quality as well as adapter-contaminated sequences were discarded. High quality (paired and unpaired) reads were assembled de-novo using SOAPde-novo assembler independently at eight different K-mers (21, 27, 33, 39, 45, 51, 57, 63). The eight assemblies were subsequently clustered by using CD-HIT-EST [68]. The clustering parameters used were ≥80% query coverage and ≥80% identity. To further clean the data transcripts present in only one of the K-mer assemblies were removed. This was followed by removal of transcripts with less than 1 FPKM in all the three conditions (BC, BDS and BHS). Finally all the transcripts less than 200 bp were removed and the remaining transcripts were functionally annotated using FASTAnnotater tool (http://fastannotator.cgu.edu.tw/) with an e-value cut-off of 0.00001 by taking non-redundant protein database of EMBL (European Molecular Biology Laboratory) as a reference. Gene ontology analysis of transcripts was derived through Uniprot hit accessions and prediction of biochemical pathways was performed by KEGG identifiers (http://www.genome.jp/kegg/).
Supplementary_files_format_and_content: cufdiff_isoform_exp.diff: List of of deferentially expressed transcripts
Supplementary_files_format_and_content: matrix_isoforms.fpkm_tracking: Normalized FPKM values of each sample (Q1=BC, Q2=BDS, Q3=BHTS)

Submission date

Dec 16, 2014

Last update date

May 15, 2019

Contact name

Gopal Joshi

E-mail(s)

gopal_joshii@yahoo.com

Phone

+91-11-27662609

Organization name

Delhi University