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Sample GSM2467253

Query DataSets for GSM2467253

Status

Public on Nov 26, 2018

Title

PC_20M

Sample type

SRA

Source name

Parietal Cortex

Organism

Macaca mulatta

Characteristics

age: 20 years
tissue: brain
tissue region: Parietal Cortex
Sex: Male

Treatment protocol

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.

Growth protocol

[animal protocol] Eight socially-housed healthy rhesus macaques used in this study were selected at random and were equally divided into two groups according to age (10 and 20 years respectively). Each group included 4 monkeys, with 2 males and 2 females. These two 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.

Extracted molecule

total RNA

Extraction protocol

For each sample, 10μg of total RNA was used for cirRNA-seq library preparation.Total RNA was treated with RQ1 DNase (Progema) to remove genomic DNA. A large proportion of mRNA was captured by oligo dT(25), and remaining RNA was purified with Ampure XP Beads. Ribosomal RNA was depleted away by RiboMinus Kit. Linear RNA (including residual rRNA, residual mRNA, and so on) was digested by RNase R.
CircRNA was iron fragmented at 95℃ followed by end repair and 5’ adaptor ligation. Then reverse transcription was performed with RT primer harboring 3’ adaptor sequence and randomized hexamer. The cDNAs were purified and amplified, then PCR products corresponding to 300~500bp were selectively captured and quantified. Libraries were stored at -70 C until used for sequencing. For high-throughput sequencing, the libraries were prepared following the manufacture’s instructions and applied to Illumina NextSeq500 system with 150x2 paired-end type by ABlife. Inc (Wuhan, China).

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

Rnase R treated circular RNA

Data processing

After reads were obtained from sequencing. Cutadapt (Martin, 2011) was used to trim adaptors (with 10% error rate) and low quality bases (less than 16). Reads that were less than 16nt in both ends were discarded.
Before prediction, we used bwa mem (Li and Durbin, 2010) method to align reads to the Rhesus Macaque (Rhesus Macaque Genome et al., 2007) reference genome in local mode (with parameters: -k 19 -T 19).
After alignment, CIRI software (Hoffmann et al., 2014) was used to predict circRNAs in macaque brain samples. Candidate circRNAs with no less than two head-to-tail junction reads were preserved.
After obtaining the circRNAs, TPM was calculated for each circRNA candidate. CircRNAs are constituted by two parts by CIRI: head-to-tail junction part and non-junction part. We set head-to-tail junctions reads as S, non-junction reads as N, and total circRNAs reads in each sample as T. The TPM value was calculated by the following formula: TPM = (S*2 + N)*1000000/T
Genome_build: MMUL 1.0
Supplementary_files_format_and_content: tab-delimited text file includes circRNA TPM values for each Sample

Submission date

Jan 24, 2017

Last update date

May 15, 2019

Contact name

dong chen

Organization name

ablife

Street address

GaoXin 2nd Road

City

wuhan