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Sample GSM3462525

Query DataSets for GSM3462525

Status

Public on Jan 01, 2020

Title

SU5402_early_3h_1

Sample type

SRA

Source name

Mix of whole embryos

Organism

Branchiostoma lanceolatum

Characteristics

treatment: SU5402 50microM
time of treatment: 5hpf
developmental stage: 8hpf

Treatment protocol

Embryos were treated with 0,1% DMSO or 50µM SU5402.

Growth protocol

Embryos were grown in 6cm Petri dishes filled with 6mL of filtered sea water with DMSO or SU5402.

Extracted molecule

total RNA

Extraction protocol

Embryos were frozen in liquid nitrogen after getting rid of the filtered sea water by centrifugation. Total RNA was extracted using the RNeasy Mini Plus Kit from QIAGEN following manufacturer's instructions. Disruption and homogenization were undertaken using TissueLyzer from QIAGEN.
A library of template molecules suitable for high throughput DNA sequencing was created following the Illumina “Truseq RNA sample preparation low throughput” protocol with some modifications. Briefly, mRNA was purified from 2 µg total RNA using oligo-dT magnetic beads and fragmented using divalent cations at 94°C for 8 minutes. The cleaved mRNA fragments were reverse transcribed to cDNA using random primers, then the second strand of the cDNA was synthesized using Polymerase I and RNase H. The next steps of RNA-Seq library preparation were performed in an automated system using SPRIworks Fragment Library System I kit (ref A84801, Beckman Coulter, Inc) with the SPRI-TE instrument (Beckman Coulter, Inc). Briefly, in this system double stranded cDNA fragments were blunted, phosphorylated and ligated to indexed adapter dimers, and fragments in the range of ~200-400 bp were size selected. The automated steps were followed by PCR amplification (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 15 cycles (6 hpt libraries) or 12 cycles (3 and 9 hpt libraries); 5 min at 72°C), then surplus PCR primers were removed by purification using AMPure XP beads (Agencourt Biosciences Corporation). DNA libraries were checked for quality and quantified using 2100 Bioanalyzer (Agilent).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

SU5402 50µM

Data processing

The first 50 bases of pass-filter reads were retained, in order to be comparable between the different samples.
These reads were mapped onto the reference transcriptome (https://wwwphp.obs-banyuls.fr/repository/transcriptome_B_lanceolatum/TotalMergeAssembliesGenbankRenomme.fasta.gz) using bwa v0.6.1 29 and the following set of parameters : -l 27 -n 4 -e 4. Only uniquely aligned reads were then retained. The number of reads aligned to each contig was computed using R.
Genome_build: Reference transcriptome constructed using data from Oulion et al (PLoS One. 2012; 7(5): e36554) and the data obtained in this study, available on the following URL : https://wwwphp.obs-banyuls.fr/repository/transcriptome_B_lanceolatum/TotalMergeAssembliesGenbankRenomme.fasta.gz
Supplementary_files_format_and_content: Tabulated text file containing raw read counts in each sample.

Submission date

Nov 07, 2018

Last update date

Jan 01, 2020

Contact name

Stephanie Bertrand

E-mail(s)

stephanie.bertrand@obs-banyuls.fr

Organization name

CNRS/SU UMR7232

Street address

AVENUE PIERRE FABRE