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Sample GSM3679471

Query DataSets for GSM3679471

Status

Public on Dec 30, 2019

Title

8_37 RNA-seq

Sample type

SRA

Source name

medulla oblongata

Organism

Macaca mulatta

Characteristics

macaque id: Rhesus 8
tissue: brain
gender: male
age: 3.8 years

Extracted molecule

total RNA

Extraction protocol

RNA-seq:RNA degradation and contamination was detected by 1% agarose gels; Then, RNA purity was checked using the kaiao K5500® Spectrophotometer (Kaiao, Beijing, China). The RNA integrity and concentration was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). ATAC-seq:A total amount of 10,000 cells at 500g for 5 min, cells were lysed using cold lysis buffer. Nuclei were spun at 500g for 10 min using a refrigerated centrifuge. The pellet was resuspended in the transposase reaction mix for 30 min at 37 °C, sample was purified using a Qiagen MinElute kit.
RNA-seq:A total amount of 3 ?g RNA per sample was used as initial material for the RNA sample preparations. Ribosomal RNA was removed using Epicentre Ribo-Zero? Gold Kits(Human/Mouse/Rat) (Epicentre, USA). Subsequently, the sequencing libraries were generated following manufacturer recommendations with varied index label by NEBNext® Ultra? Directional RNA Library Prep Kit for Illumina (NEB, Ispawich, USA) . ATAC-seq: The libraries were purified using a Qiagen PCR cleanup kit yielding a final library concentration of ~30 nM in 20 ?L. Libraries were amplified for a total of 10?15 cycles. Libraries were sequenced on Illumina NextSeq 500 (Next500 kit v2 High Output 150 cycles).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Description

Rhesus_RNA_gene_count.txt

Data processing

RNA-seq: The fastq files generated by the Illumina workflow underwent read-level quality control before mapping, using FastQC (v0.11.4). For sample with poor read quality, quality trimming was performed using Trim Galore (v0.4.4, Quality Phred score cut-off: 30).The HISAT2 (v2.0.5, default parameters) was utilized for mapping the reads with the Mmul 8.0.1 reference genome and the Mmul 8.0.1.91 transcriptomic annotation GTF. Read counts of genes were generated using HTSeq (v0.6.1).
ATAC-seq: paired-end reads were aligned to the rhesus macaque genome (Mmul 8.0.1, downloaded from ENSEMBL), using bwa mem (v0.7.12) with -M parameter. First, PCR duplicates were removed using Picard (v1.119). Reads mapped to mitochondrial DNA were then excluded. Only uniquely mapped and properly paired reads with insert size less than 2kb and mapping quality over 30 were kept for downstream analysis. UCSC bedGraphToBigWig was used to generate bigWig files. Peak calling was performed with MACS2 (v2.1.0.20151222) using parameters ?--nomodel --nolambda --shift 100 --extsize 200 -B --SPMR?.
Genome_build: Mmul 8.0.1
Supplementary_files_format_and_content: tab-delimited text files include count values for each Sample; bigwig

Submission date

Mar 19, 2019

Last update date

Dec 30, 2019

Contact name

keying Lu

E-mail(s)

[email protected]

Organization name

Sichuan university

Department

State Key Laboratory of Biotherapy