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| Status |
Public on Oct 31, 2021 |
| Title |
PDR S2 |
| Sample type |
SRA |
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| Source name |
RNV
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| Organism |
Homo sapiens |
| Characteristics |
tissue: RNV
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| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue samples were stored in RNAlater (Qiagen) at 2-8° until sequencing was performed. RNA extraction, library preparation and RNA sequencing were conducted at the Genomics Core Facility “KFB - Center of Excellence for Fluorescent Bioanalytics” (University of Regensburg, Germany; www.kfb-regensburg.de) as previously described (Boeck et al., 2020). In brief, total RNA was extracted from RNV and ILM tissue samples and stabilized in RNAprotect Cell Reagent according to the RNeasy Plus Micro Kit protocol (Qiagen). After pelleting, the RNAprotect buffer was removed, replaced by RLT Plus buffer and the samples were homogenized by vortexing for 30 sec. Genomic DNA contamination was eliminated using gDNA Eliminator spin columns. Next, ethanol was added and the samples were applied to RNeasy MinElute spin columns followed by several wash steps. Finally, total RNA was eluted in 12 μl of nuclease-free water.
RNA sequencing analysis was performed for 24 samples. The SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories) was used to generate first-strand cDNA from 750 pg total-RNA. Double-stranded cDNA was amplified by LD PCR (12 cycles) and purified via magnetic bead clean-up. Library preparation was carried out, as described in the Illumina Nextera XT Sample Preparation Guide (Illumina). 150 pg of input cDNA was 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 index 1 (i7) and index 2 (i5) primers were used. The libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems). Equimolar amounts of each library were pooled, and the pools then used for cluster generation on the cBot with the Illumina TruSeq SR Cluster Kit v3. The sequencing run was performed on a HiSeq 1000 instrument using the indexed, 50 cycles single-read (SR) protocol and 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 bcl2fastq v2.18 software.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1000 |
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| Data processing |
Quality control FastQC Galaxy Version 0.72
Reads were mapped to the human reference genome (Gencode, release 35, https://www.gencodegenes.org/human/releases.html) with RNA STAR (Galaxy Version 2.7.5b, default parameters) using the Gencode annotation file (Gencode, Release 35).
Three BAM files for each sample (one for each lane) were combined in one BAM file per sample using Merge BAM files (Galaxy Version 1.2.0).
Reads mapped to the human reference genome were counted via featureCounts (Galaxy Version 1.6.4, default parameters) using the aforementioned annotation file.
The output of featureCounts was imported to RStudio (version 1.4.1103, R Version 4.0.3).
Genesymbols and genetypes were determined based on Ensembl (Release 101, download: 28.10.2020). Genes with 0 reads in all samples were removed from further analysis. Principal component analysis (PCA) was applied to check for potential batch effects. Normalized reads and differential gene expression were calculated using the R package DESeq2 (version 1.30.1) with default parameters (Benjamini-Hochberg adjusted p-values.
Genome_build: hg38
Supplementary_files_format_and_content: normalized reads
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| Submission date |
Jul 06, 2021 |
| Last update date |
Oct 31, 2021 |
| Contact name |
Clemens Lange |
| Organization name |
Uniklinik Freiburg
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| Department |
Klinik für Augenheilkunde
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| Street address |
Kilianstraße 5
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| City |
Freiburg |
| ZIP/Postal code |
79106 |
| Country |
Germany |
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| Platform ID |
GPL15433 |
| Series (1) |
| GSE179568 |
In-depth molecular characterization of neovascular membranes suggests a role for hyalocytes-to-myofibroblasts transdifferentiation in proliferative diabetic retinopathy |
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| Relations |
| BioSample |
SAMN20081559 |
| SRA |
SRX11362050 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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