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Sample GSM2098624

Query DataSets for GSM2098624

Status

Public on Apr 15, 2016

Title

48hpf_con1 (ESD-13_N1)

Sample type

SRA

Source name

heart

Organism

Danio rerio

Characteristics

line: AB
tissue: heart
developmental stage: 48hpf

Growth protocol

Embryos were collected, staged and grown under standard conditions

Extracted molecule

total RNA

Extraction protocol

Dissected hearts were lysed and the RNA extracted using a Nucleospin XS kit from Machinery Nagel, according to the manufacturers instructions
Amplified cDNA was prepared from 5 ng of total RNA using the Ovation RNA-seq system V2 (NuGEN Technologies, Inc.) following manufacturer's instructions. Briefly, total RNA were reverse-transcribed into first-strand cDNA using a combination of random and poly-T DNA/RNA chimeric SPIA primers. Priming sites created by a heating fragmentation of mRNA within the cDNA/mRNA complex were then used to synthesize the second strand cDNA using a DNA polymerase. The resulting double-stranded cDNA with a unique RNA/DNA heteroduplex at one end were purified using Agencourt RNAClean XP beads (Beckman Coulter Inc.) and used as substrate in the linear amplification process, SPIA (single primer isothermal amplification). Amplified cDNA was purified using AMPure XP beads (Beckman Coulter Inc.) and 500 ng was fragmented by sonication using a Covaris E210 instrument (with duty cycle: 10X, intensity: 5 and cycle/burst: 200 for 180 seconds). The next steps of RNA-Seq Library preparation were performed on the Mondrian™ SP Workstation using Ovation® SP Ultralow Library Systems kit (NuGEN Technologies, Inc.) according to manufacturer's instructions. Briefly, 100 ng of amplified cDNA were blunted, phosphorylated and ligated to indexed adapter dimers. The libraries were then enriched by PCR amplification (2 min at 72 degrees Celsius; [30 sec at 94 degrees Celsius, 30 sec at 60 degrees Celsius, 1 min at 72 degrees Celsius] x 8 cycles; 5 min at 72 degrees Celsius) and surplus PCR primers were removed by purification using AMPure XP beads. DNA libraries were checked for quality using 2100 Bioanalyzer (Agilent) and quantified using Kapa Sybr Fast Light Cycler 480 qPCR Kit (Kapa Biosystems) according to manufacturer's recommendations. The libraries were loaded in the flow cell at 7pM concentration and sequenced in the Illumina Hiseq 2500 as single-end 50 base reads following Illumina’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Image analysis and base calling were performed using RTA 1.17.21.3 and CASAVA 1.8.2.
Reads were mapped onto the zv9 assembly of Danio rerio genome using Tophat2.0.10 and bowtie-2-2.1.0.
Quantification of gene expression has been performed using HTSeq-0.5.4p3.
Normalization was performed using DESeq_1.10.1.
Genome_build: zv9
Supplementary_files_format_and_content: tabulated text file containing normalized read counts.

Submission date

Mar 24, 2016

Last update date

May 15, 2019

Contact name

Julien Vermot

E-mail(s)

julien@igbmc.fr

Organization name

IGBMC

Street address

1 rue Laurent Fries