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| Status |
Public on Jul 06, 2017 |
| Title |
CB 4F cage_seq |
| Sample type |
SRA |
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| Source name |
4-yr old female brain
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| Organism |
Macaca mulatta |
| Characteristics |
tissue: cerebellar cortex
age: 4 year old
gender: female
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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).
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% Agrose gel electrophoresis. For each sample, 10μg of total RNA was used for CAGE-seq library preparation. Polyadenylated mRNAs were purified and concentrated with oligo(dT)-conjugated magnetic beads (invitrogen) .In brief, mRNA was treated with T4 polynucleotide kinase (NEB) at 37℃ for 30min and purified with oligo(dT)-conjugated magnetic beads (invitrogen). and subsequently degested with Terminator™ 5´-Phosphate-Dependent Exonuclease (Ambion) at 30℃ for 30min and purified with oligo(dT)-conjugated magnetic beads (invitrogen). The capped mRNA was performed with RT primer， then synthesized DNA with Terminal-Tagging oligo. The cDNAs were purified and amplified with PCR primers (illumina) and PCR products corresponding to 200-500 bps were purified, quantified and stored at -80 ℃ until used for sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
CAGE |
| 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.
The end first reads for paired sequencing was used to do alignment and transcription start site identification. After alignment 5 prime end of each reads was considered as the CAGE tag-defined transcription start sites (CTSSs). The number of CAGE tags mapping to each CTSS across different samples was normalized to obtain the normalized number of tags per million (tpm).
We then combined the TSSs to transcription clusters (TCs) according the known method (Nepal et al., 2013). Only CTSSs supported by a minimum of 0.5 tpm in at least one sample were used for a sample-specific clustering into transcript clusters (TCs). Neighboring CTSSs were clustered if they were <20 bp apart.
Genome_build: MMUL 1.0
Supplementary_files_format_and_content: tabular data of text format,the TSS TPM value of each sample
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| Submission date |
Sep 21, 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) |
| GSE87176 |
Spatiotemporal- and sex-specific Dynamics of lncRNA expression links to brain development and aging in Rhesus Macaque (CAGE-seq) |
| GSE87182 |
Annotation and cluster analysis of spatiotemporal- and sex-related lncRNA expression in Rhesus macaque brain |
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| Relations |
| BioSample |
SAMN05799236 |
| SRA |
SRX2182935 |
| 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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