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| Status |
Public on Jan 28, 2023 |
| Title |
CX3 3013 |
| Sample type |
SRA |
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| Source name |
Retina
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| Organism |
Mus musculus |
| Characteristics |
tissue: Retina
genotype: WILD-TYPE
treatment: 6wks Diabetic
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from enucleated retinas using the Qiagen RNeasy Kit following tissue homogenization using Zymo Research BashingBead lysis tubes.
Approximately 500ng of total RNA was used for stranded mRNA-seq library preparation by following the NEB Directional mRNA-seq sample preparation guide. The first step in the workflow involved purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cautions under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Strand specificity was achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM). These cDNA fragments then went through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. The products were then purified and enriched with PCR to create the final RNA-seq library. Finally RNA-seq libraries were subjected to quantification process, pooled for cBot amplification and subsequent 100bp paired read sequencing run with Illumina NovaSeq 6000 platform.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
3013_S25
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| Data processing |
After the sequencing run, demultiplexing with Bcl2fastq2 was employed to generate the fastq file, with the average of 30M reads per sample
Raw reads were imported into CLC Genomics Workbench v21.0.5
Initially, adaptors were trimmed and remaining reads for each sample were mapped to the annotated mouse genome (GRCm39), followed by differential gene expression (DEG) analysis using the RNA-seq analysis tools within the CLC genomics software
Assembly: GRCm39
Supplementary files format and content: excel file outlining the differentially expressed genes in the comparison listed in the file name
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| Submission date |
Jan 23, 2023 |
| Last update date |
Jan 28, 2023 |
| Contact name |
Kaira Adriana Church |
| Organization name |
University of Texas at San Antonio
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| Department |
Molecular Microbiology and Immunology
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| Lab |
Astrid Cardona
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| Street address |
One UTSA Circle 1.212
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| City |
San Antonio |
| State/province |
TX |
| ZIP/Postal code |
78249 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE223522 |
Effects of transient microglia depletion and repopulation on retinal gene expression in the diabetic murine retina |
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| Relations |
| BioSample |
SAMN32877621 |
| SRA |
SRX19143455 |
| 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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