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Sample GSM3511132

Query DataSets for GSM3511132

Status

Public on Aug 31, 2020

Title

BTLT4_S2Hoxb1Mu

Sample type

SRA

Source name

Amplified ds cDNA with Ovation V2 Nugen kit

Organism

Mus musculus

Characteristics

tissue: second heart field region
Stage: E8.5
genotype: Hoxb1Mu

Extracted molecule

polyA RNA

Extraction protocol

SHF was microdissected, flash frozen on dry ice, and RNA was harvested using the NucleoSpin® RNA XS kit
Amplified cDNA was prepared from 2 ng of total RNA using the Ovation RNA-seq system V2 (NuGEN Technologies, Inc.) following manufacturer's instructions. Briefly, first-strand cDNA was prepared from total RNA using a combination of random and poly-T DNA/RNA chimeric SPIA primers and reverse transcriptase, resulting in a cDNA/mRNA hybrid molecule. Priming sites created by a heating fragmentation of the mRNA within the cDNA/mRNA complex were 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 was purified using Agencourt RNAClean XP beads (Beckman Coulter Inc.). The purified product was then used as the substrate of a linear amplification process, SPIA (single primer isothermal amplification). During the amplification step, RNase H degrades RNA in DNA/RNA heteroduplex at the 5'end of the first cDNA strand, creating a binding site for the DNA/RNA SPIA chimeric primer, then DNA polymerase starts replication at the 3'end of the primer displacing the existing forward strand. The process including SPIA DNA/RNA primer binding, DNA replication, strand displacement and RNA degradation is repeated and results in rapid generation of microgram quantities of amplified cDNA. 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 using Ovation Ultralow Library System kit (NuGEN Technologies, Inc.) according to manufacturer's instructions. Briefly, in this system 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 6 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) following manufacturer'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 2.7.3 and bcl2fastq 2.17.1.14.
RNA-Seq reads were aligned using the STAR aligner version 2.5.2
Transcripts abundance was computed using HTSEQ-count 0.9.12 and the background was estimated through read counts from intergenic regions using windows of 5 kb length
Normalisation and differential gene expression analysis between conditions were performed using R (R version 3.3.4) and DESEQ2
Genome_build: mm10
Supplementary_files_format_and_content: The final processed data files is the output of the DESEQ2 package. This file contains the gene name, the base mean , the log2foldchange, the test value, the p-value, the adjusted p-value and the normalized count foreach sample.

Submission date

Dec 13, 2018

Last update date

Aug 31, 2020

Contact name

Sonia Stefanovic

E-mail(s)

Sonia.STEFANOVIC@univ-amu.fr

Organization name

INSERM U1251

Street address

27 BD JEAN MOULIN