|
| Status |
Public on May 15, 2024 |
| Title |
PFA_Retina_rep3 |
| Sample type |
SRA |
| |
|
| Source name |
Retina
|
| Organism |
Canis lupus familiaris |
| Characteristics |
tissue: Retina
background breed: Beagle
age: 31 weeks
Sex: Male
genotype: wild-type
treatment: PFA-fixed, frozen
|
| Treatment protocol |
The left eyes were fixed in 4% PFA in 0.1 M PBS for 3 hours, followed by an additional 24-hour fixation in 2% PFA in 0.1 M PBS at 4°C. Subsequently, the eyecup was dissected into four quadrants, and the tissues were cryoprotected sequentially for 24 hours each in 15% and 30% sucrose solutions in 0.1 M PBS at 4°C. After cryopreservation, the PFA-fixed tissues were also embedded in optimal cutting medium (OCT) medium, following rapid freezing.
For the right eye, the eyecup was dissected into four quadrants and the tissue sections embedded in OCT medium without fixation and cryoprotection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from FF and PFA-fixed samples using the RNeasy Plus Micro kit and the RNeasy FFPE kit, respectively.
For the PFA-fixed samples, specific modifications were made to the manufacturer’s protocol. The solubilized tissue sections were incubated with Proteinase K for 18 hours at 56°C for improved RNA extraction efficiency, and a second round of genomic DNA digestion was performed using the DNA-free™ DNA Removal kit post RNA extraction to ensure complete removal of genomic DNA contamination.
Sequencing libraries were constructed from RNA extracted from 4 FF and 4 PFA-fixed tissue samples using the SMARTer Stranded Total RNA-Seq kit v3 - Pico Input Mammalian.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
| |
|
| Data processing |
The fastq files were pseudo-aligned to the canine reference transcriptome (ROS_Cfam_1.0) and transcripts per million (TPM) was calculated using kallisto version 0.48.0.
The quantification data were summarized to genes employing the tximport package and normalized using the trimmed mean of M values (TMM) method in edgeR in R.
Genes with <1 count per million (CPM) across all groups were filtered out.
The normalized filtered data underwent variance stabilization through the voom function in the limma package in R.
Differentially expressed genes (³2-fold and £5% false discovery rate) were identified with linear modeling using limma after correcting for multiple testing using Benjamini-Hochberg.
Assembly: ROS_Cfam_1.0
Supplementary files format and content: Tab-delimited text file includes log2 CPM values for each sample.
|
| |
|
| Submission date |
Mar 27, 2024 |
| Last update date |
May 15, 2024 |
| Contact name |
Raghavi Sudharsan |
| E-mail(s) |
raghavi@vet.upenn.edu
|
| Organization name |
University of Pennsylvania
|
| Street address |
3900 Delancey Street
|
| City |
Philadelphia |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL33053 |
| Series (1) |
| GSE262625 |
An optimized workflow for transcriptomic analysis from archival paraformaldehyde-fixed retinal tissues collected by laser capture microdissection |
|
| Relations |
| BioSample |
SAMN40632453 |
| SRA |
SRX24080446 |
