|
| Status |
Public on Apr 12, 2023 |
| Title |
72H |
| Sample type |
SRA |
| |
|
| Source name |
orbitofrontal cortex
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague Dawley
Sex: male
age: adult
treatment: 10 days of heroin self-administration (0.03mg/kg)
tissue: orbitofrontal cortex
|
| Treatment protocol |
Adult male Sprague Dawley rats were aged 8 weeks on arrival. Forheroin exposure, animals were acclimited to the animal facility for 1 week then underwent jugular vein catheterization. Following 1 week of recovery, animals self-administered heroin at a low dosage (HL-0.03mg/kg/infusion) or a high dosage (HH- 0.075mg/kg/infusion) for 6 hours per day in 10 daily sessions. Naive animals were briefly handled each day. Following the last self-administration session, animals remained in their homecage for 21 days. After 21 days, animals were euthanized and their brains were frozen in ice cold isopentane. Orbitofrontal cortex tissue was dissected from each animal on dry ice then RNA extraction proceeded.
|
| Growth protocol |
N/A
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the miRvana PARIS kit (AM1556) from Invitrogen according to the manufacturer's protocol.
Small-RNA sequencing was performed on four biological replicates per group by BGI Genomics (BGI Americas Corp, Cambridge, MA, USA). Total RNA was assessed for quality before library preparation and RNA Integrity Numbers (RIN) were ≥ 7.5 with 28S/18S ≥ 1.3. Small RNAs (18-30 nt) were separated from total RNA by PAGE gel. Selected small RNAs were ligated with 3’ and 5’ adapters. The formed strands were reverse transcribed in cDNA and PCR amplified with high-ping polymerase. The PCR products (100-120bp) were separated by PAGE gel to eliminate primer-dimers and other byproducts and purified. The PCR yield was quantified followed by single strand DNA cyclization (ssDNA circle) for final library construction. DNA nanoballs (DNBs) were generated by ssDNA circle by rolling circle replication. The DNBs were then loaded onto the BGISEQ-500 platform sequencing for 100bp paired end reads.
|
| |
|
| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
BGISEQ-500 |
| |
|
| Data processing |
Basecalling was performed with BGI proprietary software
Raw sequencing reads were filtered to remove reads with low quality tags, 5’ primer contaminants, tags without a 3’ primer, tags with poly A and those shorter than 18nt and the remaining reads were stored in FASTQ format.
Bowtie2 was used to align clean reads to the reference genome (rn6).
miRDeep2 was used to predict novel miRNA based on harpin structure of miRNA precursors.
miRNAs expression level was calculated as Transcripts Per Kilobase Million (TPM) to directly compare differences in gene expression between samples.
Assembly: rn6
Supplementary files format and content: raw counts
|
| |
|
| Submission date |
Jun 03, 2022 |
| Last update date |
Apr 12, 2023 |
| Contact name |
Stephanie Daws |
| E-mail(s) |
stephanie.daws@temple.edu
|
| Organization name |
Temple University
|
| Street address |
3500 N Broad St
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19140 |
| Country |
USA |
| |
|
| Platform ID |
GPL24782 |
| Series (1) |
| GSE205451 |
small-RNA sequencing after forced abstinence from heroin self-administration in male rats |
|
| Relations |
| BioSample |
SAMN28860733 |
| SRA |
SRX15590268 |
