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| Status |
Public on Apr 05, 2022 |
| Title |
spinal cord normal saline 7days rep2 IP |
| Sample type |
RNA |
| |
|
| Source name |
spinal cord
|
| Organism |
Rattus norvegicus |
| Characteristics |
tissue: spinal cord
group: saline
antibody: anti-N6-methyadenosine (m6A)
|
| Treatment protocol |
The adult male SD rats were randomly divided into normal saline group (NS group) and morphine tolerance group (MT group). Rats in the MT group were administrated morphine hydrochloride injection 10 μg/10 μl intrathecally through the tube inserted into the sheath at 8:00 a.m. and 20:00 p.m. for 7 consecutive days, the NS group was given 10 μl of normal saline at the same time point.
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| Growth protocol |
The animals were housed in a specific pathogen-free and individually ventilated cages at a room with 12/12 light-dark cycle and temperature of 22 ± 1 °C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The total RNA of rat lumber spinal cords (L4 - L6) was extract following the standard protocol of TRI Reagent.
|
| Label |
Cy5
|
| Label protocol |
The total RNAs were immunoprecipitated with anti-N6-methyadenosine (m6A) antibody. The modified RNAs were eluted from the immunoprecipitated magnetic beads as the “IP”. The unmodified RNAs were recovered from the supernatant as “Sup”. The “IP” and “Sup” RNAs were treated with RNase R, and then labeled with Cy5 and Cy3 respectively as cRNAs in separate reactions using Arraystar Super RNA Labeling Kit.
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| |
|
| Hybridization protocol |
The labeled cRNAs were combined and hybridized to Arraystar Rat CircRNA Epitranscriptomic Arrays (8x15K, Arraystar) at 65 °C for 17 hours in an Agilent Hybridization Oven.
|
| Scan protocol |
The arrays were scanned in two-color channels by an Agilent Scanner G2505C (Agilent, the USA) after washing.
|
| Description |
Biological replicate 2 of 4. spinal cords of normal rats
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| Data processing |
The raw RNA intensities of IP (Cy5-labelled) and Sup (Cy3-labelled) were analyzed by Agilent Feature Extraction software (version 11.0.1.1) and normalized with average of log2-scaled spike-in RNA intensities. After Spike-in normalization, the probe signals having Present (P) or Marginal (M) QC flags in at least 4 out of 8 samples were retained for further “m6A methylation level” analyses. “m6A methylation level” was calculated for the percentage of modification based on the IP (Cy5-labelled) and Sup (Cy3-labelled) normalized intensities. Differentially m6A-methylated circRNAs between two comparison groups were identified by filtering with the fold change and statistical significance (p-value) thresholds.
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| |
|
| Submission date |
Mar 31, 2022 |
| Last update date |
Apr 06, 2022 |
| Contact name |
Manyu Xing |
| E-mail(s) |
xmy0371@csu.edu.cn
|
| Organization name |
Xiangya hospital, Central South University
|
| Street address |
87 Xiangya Road
|
| City |
Changsha |
| State/province |
Hunan |
| ZIP/Postal code |
410008 |
| Country |
China |
| |
|
| Platform ID |
GPL29537 |
| Series (1) |
| GSE199892 |
Epitranscriptomic map of N6-methyladenosine circular RNAs in the spinal cord of morphine-tolerant rats |
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