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| Status |
Public on Nov 01, 2016 |
| Title |
Sham surgery control |
| Sample type |
SRA |
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| Source name |
RGC TRAP
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| Organism |
Xenopus laevis |
| Characteristics |
tissue: left eye
time (days post-surgery): 1
treatment: control (sham surgery)
cell type: RGC
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| Treatment protocol |
The retinas from each of 10 post-metamorphic transgenic Tg(Islet2b:EGFP-RPL10a) Xenopus laevis frogs, 3.5 - 5.0 cm in length, were either left untreated (naïve) or received a surgery (operated). Operated individuals were anesthetized with 0.05% tricaine methanesulfonate and received either a sham surgery (sham) or a crush injury to the right optic nerve (crush), and no treatment the left optic nerve (control). To avoid the surrounding vasculature, an initial surgical incision was made in the right medial roof of the buccal cavity, the muscles were then gently teased apart and the optic nerve was exposed from at a 45o angle. The exposed nerve was gently separated from the adjacent ophthalmic artery and a pair of #55 forceps were used to perform a five second crush located 5 mm from the optic nerve head. Crush injuries were verified by visual inspection by observing a region of clear sheath flanked by a more opaque area. Frogs with excessive bleeding were excluded from further study. Following the surgery, frogs were allowed to recover in a shallow bath of 0.1X MMR (10 mM NaCl, 0.2 mM KCl, 0.1 mM MgCl2, 0.2 mM CaCl2, 0.5 mM HEPES; pH 7.5), transferred to individual tanks, and immersed in fresh 0.1 X MMR. Ten untreated (naïve) frogs were also moved to individual tanks for a day. At 24 hours post-surgery, frogs were transferred to 18.25" x 12" x 6.25"communal tanks with a maximum of ten frogs per tank. The surgically manipulated frogs were allowed to recover for 1, 3, 7, or 11 days post optic nerve crush. Following the crush surgery at these discreet time periods, the frogs were immersed in 0.5% tricaine methanesulfonate, decapitated and exsanguinated.
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| Growth protocol |
Restriction enzyme mediated integration was used to create lines of Xenopus laevis that stably express TRAP transgenes in RGCs . Biologically independent experiments were each carried out using F1 progeny from single tg(islet2b:EGFP-RPL10a) female founder lines. These progeny were screened for presence of the EGFP-rpl10a transgene using a fluorescent dissecting microscope. eGFP positive progeny were then grown to post-metamorphic stage (> 6 months) under 12L:12D photoperiod at 22 oC. All animal experiments were carried out using procedures approved by the Washington and Lee University and Johns Hopkins University School of Medicine’s IACUCs.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Both left and right eyes were harvested separately. Retinas were isolated by removing the lens, then peeling off and discarding the retinal pigment epithelial layer. Freshly dissected retinas were immersed in ice-cold lysis buffer (20 mM HEPES KOH, pH 7.4, 5 mM MgCl2, 150 mM KCl) to which was added freshly prepared 100 μg/ml cycloheximide, 0.5 mM DTT, protease inhibitors (Roche Mini Complete, EDTA-Free) and 40 U/ml recombinant Rnasin (Promega). We isolated total RNA from the retina or used the translation ribosomal affinity purification (TRAP) protocol to isolate and purify the RNAs specific to the RGCs (described in Watson et al., 2012; DOI 10.1002/dvdy.23880). To immunoprecipitate ribosomes and associated RNAs, we used equal amounts of anti-eGFP antibodies (19C8 and 19F7; Memorial Sloan-Kettering Monoclonal Antibody Facility) at a concentration of 100 µg of total anti-eGFP antibody per 375 µl of Dynal Protein G-magnetic beads (Life Technologies).
To obtain a minimum of 120 ng/sample of purified TRAP-isolated mRNAs, equal quantities from each of the three independent biological replicates were combined to generate a single pooled sample. RNA samples were then sent to the JHMI Deep Sequencing Core for RNA-Seq cDNA library construction using poly(A) selection, with standard Illumina protocols for HiSeq 2000.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
d1.shc
TRAP RNA
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| Data processing |
Raw sequencing reads in FASTQ format were filtered for quality and adapter sequences removed using Trimmomatic (v. 0.33) with the following parameters: a 4-base sliding window with average quality cutoff of 15, removal of 3 leading and trailing low quality bases and a minimum read length of 36 bases.
Surviving reads were aligned to the Xenopus laevis genome using the JGI gene models version 9.1, retrieved from xenbase.org on 11/21/2015, using Bowtie2 (v. 2.2.5) with ‘sensitive’ alignment presets augmented with a minimum acceptable alignment score of -0.1.
Gene expression estimation, expressed as fragments per killibase per million reads (FPKM), was performed using RSEM (v. 1.2.21) with default options.
Genome_build: XENLAv9.1 (X. laevis 9.1, xenbase.org)
Supplementary_files_format_and_content: .csv files represent TPMs and FPKMs
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| Submission date |
Feb 09, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Gregg Brooks Whitworth |
| E-mail(s) |
whitworthg@wlu.edu
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| Phone |
(415) 336-3872
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| Organization name |
Washington and Lee Unversity
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| Department |
Biology
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| Street address |
204 West Washington St
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| City |
Lexington |
| State/province |
VA |
| ZIP/Postal code |
24450 |
| Country |
USA |
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| Platform ID |
GPL17682 |
| Series (1) |
| GSE77724 |
Translational profiling of retinal ganglion cell optic nerve regeneration in Xenopus laevis |
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| Relations |
| BioSample |
SAMN04481656 |
| SRA |
SRX1568300 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2057923_d1.shc.csv.gz |
576.8 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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