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Sample GSM2323948 Query DataSets for GSM2323948
Status Public on Jul 06, 2017
Title OC 10M cage_seq
Sample type SRA
 
Source name 10-yr old male brain
Organism Macaca mulatta
Characteristics tissue: occipital cortex
age: 10 year old
gender: male
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.
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection CAGE
Instrument model Illumina NextSeq 500
 
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
 
Submission date Sep 21, 2016
Last update date May 15, 2019
Contact name Dong Chen
Organization name ABLife, Inc.
Department Center for Genome Analysis
Street address 388 GaoXin 2nd Road, East Lake Hi-Tech Development Zone
City Wuhan
State/province Hubei
ZIP/Postal code 430075
Country China
 
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
Relations
BioSample SAMN05799222
SRA SRX2182947

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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