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Sample GSM2701990

Query DataSets for GSM2701990

Status

Public on Mar 14, 2018

Title

Control rep2

Sample type

SRA

Source name

Retina

Organism

Mus musculus

Characteristics

strain: C57BL/6J
tissue: retina
treatment: laser treatment between vessels

Treatment protocol

During anesthesia with a mixture of 100 mg ketamine and 5 mg xylazine /kg, mice were injected i. p. with 200 mg/kg of a 100 mg/ml solution of eosin Y in 0.9 % NaCl to enhance the laser effect. The settings of the laser (Visulas 532s, Carl Zeiss Meditec, Jena, Germany) were 50 mW, spot size of 50 µm, 2.5 s duration. 3 - 6 laser applications were necessary to funduscopically occlude a vein. All veins of one eye of each mouse were occluded while the other eye served as control receiving the same laser application scheme (number of laser sites, intensity, and distance from the papilla) in retinal areas between the large vessels.

Growth protocol

12 weeks old C57BL/6J mice were used. They were housed at 24 °C with a 12 h light / dark rhythm.

Extracted molecule

total RNA

Extraction protocol

RNA was isolated from the retinas with the RNeasy kit (Qiagen, Hilden, Germany).
Library preparation and RNAseq were carried out as described in the Illumina TruSeq Stranded mRNA Sample Preparation Guide, the Illumina HiSeq 1000 System User Guide (Illumina, Inc., San Diego, CA, USA), and the KAPA Library Quantification Kit - Illumina/ABI Prism User Guide (Kapa Biosystems, Inc., Woburn, MA, USA). In brief, 300 ng of total RNA was used for purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented to an average insert size of 200-400 bases using divalent cations under elevated temperature (94 °C for 4 min). The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity was achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM), followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The incorporation of dUTP in second strand synthesis quenches the second strand during amplification, because the polymerase used in the assay is not incorporated past this nucleotide. The addition of Actinomycin D to First Strand Synthesis Act D mix (FSA) prevents spurious DNA-dependent synthesis, while allowing RNA-dependent synthesis, improving strand specificity. These cDNA fragments then had the addition of a single 'A' base and subsequent ligation of the adapter. The products were purified and enriched with PCR to create the final cDNA library. The libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems, Inc., Woburn, MA, USA). Equimolar amounts of each library were pooled, and the pools were used for cluster generation on the cBot with the Illumina TruSeq SR Cluster Kit v3. The sequencing run was performed on an HiSeq 1000 instrument using the indexed, 50 cycles single-read (SR) protocol and the TruSeq SBS v3 Reagents according to the Illumina HiSeq 1000 System User Guide. Image analysis and base calling resulted in .bcl files, which were converted into .fastq files with the CASAVA1.8.2 software.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 1000

Data processing

Read mapping: STAR 2.5.2b
Feature counting: featureCount from Subread 1.5.1 for R
Genome_build: mm11
Supplementary_files_format_and_content: tab delimited file containing gene symbols and reads for every sample

Submission date

Jul 13, 2017

Last update date

May 15, 2019

Contact name

Gottfried Martin

E-mail(s)

gottfried.martin@uniklinik-freiburg.de

Organization name

Universitätsklinikum Freiburg

Department

Klinik für Augenheilkunde