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Sample GSM7468745

Query DataSets for GSM7468745

Status

Public on Jan 03, 2024

Title

nOIR_p17_S3

Sample type

SRA

Source name

retina

Organism

Mus musculus

Characteristics

tissue: retina
strain: C57BL/6
genotype: WT
treatment: Control p17

Extracted molecule

total RNA

Extraction protocol

The fellow eye of these mice as well as both eyes pooled for other animals from that litter were processed for retinal extraction straight after intracardiac PBS perfusion and enucleation. Instead of mounting them on a template, however, they were stored in RNAlater for up to 6 days and shipped at 2-8°C to the RNA sequencing provider. Samples containing one and two retinae showed similar quality and quantity of extracted RNA. Total RNA was extracted from mouse retina stabilized in RNAlater buffer according to the “Purification of total RNA from animal and human tissue” protocol of the RNeasy Micro Kit (QIAGEN, Hilden, Germany) and was performed by the Genomics Core Facility “KFB - Center of Excellence for Fluorescent Bioanalytics” (University of Regensburg, Regensburg, Germany; www.kfb-regensburg.de). In brief, after centrifugation for 5 minutes at 5,000 x g and removing the RNAlater, the tissue was disrupted and homogenized in 350 µl RLT buffer containing 1% beta-mercaptoethanol with Precellys CK14 ceramic beads (1 cycle of 15 seconds at 5,500 rpm) using a Precellys 24 Homogenisator (Bertin Corp., Rockville, MD, USA). Next, the sample was centrifuged for 2 minutes at full speed and 350 µl of the cleared supernatant was transferred to a new tube. One volume of 70 % ethanol was added and the samples were applied to a RNeasy MinElute spin column followed by an on-column DNase digestion and several wash steps. Finally, total RNA was eluted in 14 μl of nuclease free water. Purity and integrity of the RNA was assessed on the Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip reagent set (Agilent, Palo Alto, CA, USA).
The SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories, Inc., Mountain View, CA, USA) was used to generate first strand cDNA from approximately 1 ng total-RNA. Double stranded cDNA was amplified by long distance (LD) PCR (11 cycles) and purified via magnetic bead clean-up. Library preparation was carried out as described in the Illumina Nextera XT Sample Preparation Guide (Illumina, Inc., San Diego, CA, USA). Thereby, 150 pg of input cDNA were tagmented (tagged and fragmented) by the Nextera XT transposome. The products were purified and amplified via a limited-cycle PCR program to generate multiplexed sequencing libraries. For the PCR step, 1:5 dilutions of the unique dual indexing (i7 and I5) adapters were used. The libraries were quantified using the KAPA Library Quantification Kit - Illumina/ABI Prism User Guide (Roche Sequencing Solutions, Inc., Pleasanton, CA, USA). Equimolar amounts of each library were sequenced on an Illumina NextSeq 2000 instrument controlled by the NextSeq 2000 Control Software (NCS) v1.2.0.36376, using one 50 cycles P3 Flow Cell with the dual index, single-read (SR) run parameters. Image analysis and base calling were done by the Real Time Analysis Software (RTA) v3.7.17. The resulting .cbcl files were converted into .fastq files with the bcl2fastq v2.20 software.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 2000

Data processing

Sequencing data was analyzed on the Galaxy web platform (usegalaxy.eu). Quality control was performed with FastQC (Galaxy Version 0.73, http://www. bioinformatics.babraham.ac.uk/projects/fastqc/, last access on 22nd January 2022).
Reads were mapped to the human or mouse reference genome (Gencode M28) with RNA STAR (Galaxy Version 2.7.8a, default parameters).
The BAM files for each sample from each lane were combined in one BAM file per sample using Merge BAM files (Galaxy Version 1.2.0).
Reads mapped to the human or mouse reference genome were counted by featureCounts (Galaxy Version 2.0.1, default parameters).
The output of featureCounts was imported to RStudio (version 1.4.1103, R Version 4.0.3). Gene symbols and gene types were determined based on Ensembl (Release 105, download: 28.01.2022). Genes with 0 reads in all samples were removed from further analysis. Principal component analysis (PCA) was applied to check for potential outlier and batch effects.
Normalized reads and differential gene expression were calculated using the R package DESeq2 (version 1.34) with default parameters (Benjamini-Hochberg adjusted p-values). Transcripts with log2 fold change (log2FC) >2 or <-2 and adjusted p-value <0.05 were considered as differentially expressed genes (DEG).
Assembly: Gencode M28
Supplementary files format and content: csv, normalized reads (DESeq2)

Submission date

Jun 08, 2023

Last update date

Jan 03, 2024

Contact name

Clemens Lange

Organization name

Uniklinik Freiburg

Department

Klinik für Augenheilkunde

Street address

Kilianstraße 5