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Sample GSM1496864

Query DataSets for GSM1496864

Status

Public on Dec 09, 2015

Title

13d_leaf_control_br2

Sample type

SRA

Source name

leaf control

Organism

Jatropha curcas

Characteristics

strain: Cape Verde islands
tissue: leaf

Treatment protocol

Potted 36-day-old seedlings (at the five-leaf stage) from both accessions were subjected to drought stress or continuously grown under well-watered conditions (Control). Drought stress was imposed by water withholding for a maximum period of 49 days, followed by a 7-day re-watering period.

Growth protocol

Homogeneous seeds were germinated in clean sand, and 10-day-old homogeneous seedlings were transplanted to 7.5 L pots containing a soil mixture of sand, peat and soil (3:1:1) and supplemented with a commercial fertilizer (Osmocote, Scotts, Netherlands) (5 g/pot) (N:P:K:Mg, 16:9:12:2.5). Plants were daily irrigated until the beginning of the treatments. Experiments were carried out in a greenhouse under natural photoperiod (29 Jun. - 24 Aug. 2011, Oeiras, Portugal) with a day/night temperature of 29±3 to 20±2ºC, day/night relative humidity of 39±8 to 69±4% and an average light intensity at plant level of 411±226 μmol photon m-2 s-1 (11-12 a.m.).

Extracted molecule

total RNA

Extraction protocol

Root tips and young leaves were collected along the drought assay and subsequent re-watering period. In detail, one leaf (≤2cm in length) per plant was cut with a scalpel blade from the apical tip of the plants and immediately frozen in liquid nitrogen. For root collection, plants were gently removed from the pot. Root tips (≤ 1 cm in length) were retracted with forceps from different parts of the root system and carefully brushed to remove any attached soil, the process never extended more than 30 seconds until samples were flash-frozen in liquid nitrogen. A pool of six plants for days 0 and 49, and three plants for days 13 and 52 were sampled per treatment and accession. Samples were stored at – 80ºC until further use. Before extraction the frozen material was grind in liquid nitrogen with a mortar and pestle into a fine powder. Total RNA was extracted using the RNeasy plant mini kit (Qiagen, Germany) according to the manufacturer’s instructions, however for leaf samples the lysis buffer was supplemented with 0.2% of PEG 20000, as suggested by Gehrig et al. (2000). After the extraction, samples were treated with DNAse (Turbo DNA-free Kit, AM1907, Ambion, USA) according to the manufacturer’s instructions to minimize genomic contaminants. RNA quality and quantity was evaluated using an Agilent 2100 Bioanalyzer with RNA 6000 Nano Chips, following manufacturer’s protocol; RNA integrity numbers (RIN) ranged from 8.3 - 9.9.
cDNA libraries were prepared using an initial amount of 400 ng of total RNA, and the Illumina’s TruSeqTM RNA Sample Preparation v2 protocol. Indexed barcodes were used for pooling, and sequencing was performed on an Illumina HiSeqTM 2000. To assess concentration and ensure an appropriate size distribution (between 200-570 bp) the cDNA libraries were checked using Bioanalyzer DNA 1000 chips. The sequencing run was carried out on a fully loaded flow cell with single-end 50 bp reads following manufacturer’s instructions with a loading amount of 9 pmol cDNA per lane. On average, each sample was covered by about 82 million reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Control (Day 13), total RNA from J. curcas leaves subjected to well watered conditions (control).

Data processing

Basecalls were performed using standard software supplied by Illumina for HiSeq 2000 sequencing.
Reads generated with HiSeq 2000 were mapped with NextGenMap v0.4.12 using default settings and producing bam-files.
Gene quantification was calculated with a python script 'rpkmforgenes.py' from the Sandberg laboratory (http://sandberg.cmb.ki.se/rnaseq/) at readcount and RPKM level (=reads per kilobase of exon model per million mapped reads according to Mortazavi et al. (2008)) using uniquely mapped reads.
Genome_build: Jatropha curcas genome version 4.5 as reference (Hirakawi et al., 2012) (ftp://ftp.kazusa.or.jp/pub/jatropha/).
Supplementary_files_format_and_content: Tab-delimited text files include raw read counts which were exclusively taken for differential gene expression profiling [J.curcas_readcounts.txt] and quantile-normalized RPKM values (to each value 1 RPKM was added) [J.curcas_normalized_rpkm.txt.gz].

Submission date

Sep 04, 2014

Last update date

May 15, 2019

Contact name

Christian Grumaz

Organization name

Fraunhofer IGB

Department

MBT

Street address

Nobelstr. 12