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| Status |
Public on Jul 06, 2017 |
| Title |
DG 10M RNA_seq |
| Sample type |
SRA |
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| Source name |
10-yr old male brain
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| Organism |
Macaca mulatta |
| Characteristics |
tissue: dentate gyrus
age: 10-year old
gender: male
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| Treatment protocol |
Animal protocol: Sixteen socially-housed healthy rhesus macaques used in this study were selected at random and were equally divided into four groups according to age (1, 4, 10 and 20 years respectively). Each group included 4 monkeys, with 2 males and 2 females. These four groups were euthanized by injected with excessive pentobarbital sodium (80 mg/kg, i.v.) and the monkeys’ carotid artery was cut to allow the blood drain. And then the whole brain was taken out and the corresponding brain tissue (4mm×2mm×2mm) was sampled with sterilized ophthalmic scissors and tweezers. According to a widely used macaque brain atlas and brainmaps (http://www. Brainmaps.org),the prefrontal cortex was sampled at the main sulci, the posterior cingulate cortex was sampled at the Brodmann’s area 23, the temporal cortex was sampled at the superior temporal gyrus, the parietal cortex was sampled at the middle sylvian fissure, the occipital cortex was sampled at the V1, and the cerebellar cortex was sampled at the cauda cerebelli. The hippocampus (including CA1 and dentate gyrus) was also sampled. All the collected samples were washed with RNA later solution (AM7021, Ambion, USA) and put in freezing tubes to store at liquid nitrogen temperature. All these operations above were completed inside one hour after the monkeys died. All animal procedures were in strict accordance with the guidelines for the National Care and Use of Animals approved by the National Animal Research Authority (P.R. China) and the Institutional Animal Care and Use Committee (IACUC) of the Kunming Institute of Zoology of Chinese Academy of Sciences.
All the collected samples were washed with RNA later solution (AM7021, Ambion, USA) and put in freezing tubes to store at liquid nitrogen temperature. All these operations above were completed inside one hour after the monkeys died. All animal procedures were in strict accordance with the guidelines for the National Care and Use of Animals approved by the National Animal Research Authority (P.R. China) and the Institutional Animal Care and Use Committee (IACUC) of the Kunming Institute of Zoology of Chinese Academy of Sciences.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
For RNA-Seq library, total RNA was extracted from all the brain tissue samples by using TRIzol Reagent(Ambion) following the manufacturer’s instructions. Total RNA was treated with RQ1 DNase (promega) to remove DNA. The quality and quantity of the purified RNA were determined by measuring the absorbance at 260 nm/280 nm (A260/A280) using smartspec plus (BioRad).
RNA integrity was further verified by 1.5% Agarose gel electrophoresis. For each sample, 10 μg of total RNA was used for RNA-seq library preparation. Polyadenylated mRNAs were purified and concentrated with oligo(dT)-conjugated magnetic beads (invitrogen) before used for directional RNA-seq library preparation. Purified mRNAs were ion fragmented at 95℃ followed by end repair and 5' adaptor ligation. Then mRNA reverse transcription was performed with RT primer harboring 3' adaptor sequence and randomized hexamer. The cDNAs were purified and amplified. And products corresponding to 200-500 bps were purified, quantified and stored at -80 ℃ before sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Raw reads were first discarded if containing more than 2-N bases, then reads were processed by clipping adaptor, removing low quality bases, and discarding too short reads (less than 16nt). FASTX-Toolkit (Version 0.0.13) was used to get the clean reads.
After that, clean reads were aligned to the Macaque genome (MMUL 1.0) by tophat2 (Kim et al., 2013). Aligned reads with more than one genome location were discarded due to their ambiguous location.
Uniquely localized reads were used to calculate reads number and RPKM value (RPKM represents reads per kilobase and per million) for each gene according to reads and genes genome location. Other statistical results, such as gene coverage and depth, reads distribution around start codon and stop codon, were also obtained.
LncRNAs were predicted by Cufflinks V2.2 (Trapnell et al., 2012). First transcripts of protein coding and non-coding were identified by cufflinks, and then cuffcompare was used to compare the transcripts with macaque known gene and novel transcripts were reserved as the candidate lncRNAs. Transcripts adjacent to known genes within 2000 bp were regarded as UTRs and lncRNAs’ coding potential was evaluated by cpc softwares with the default parameters. Finally, lncRNAs with multiple exons obtained from different tissue were merged together as the final lncRNA set. LncRNA prediction pipeline was described in the Sup Figures.
After getting the Expression level of all genes in all samples, differentially expressed genes were analyzed by using edgeR(Robinson et al., 2010), one of R packages. 0.01 p-value and 2 fold change were set as the threshold to define DEGs.
Genome_build: MMUL 1.0
Supplementary_files_format_and_content: [.xlsx] tabular data reporting gene RPKMs for each sample
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| Submission date |
Sep 07, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Dong Chen |
| Organization name |
ABLife, Inc.
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| Department |
Center for Genome Analysis
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| Street address |
388 GaoXin 2nd Road, East Lake Hi-Tech Development Zone
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| City |
Wuhan |
| State/province |
Hubei |
| ZIP/Postal code |
430075 |
| Country |
China |
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| Platform ID |
GPL21120 |
| Series (2) |
| GSE85377 |
Spatiotemporal- and sex-specific Dynamics of lncRNA expression links to brain development and aging in Rhesus Macaque (RNA-seq) |
| GSE87182 |
Annotation and cluster analysis of spatiotemporal- and sex-related lncRNA expression in Rhesus macaque brain |
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| Relations |
| BioSample |
SAMN05735468 |
| SRA |
SRX2143680 |
| 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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