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Sample GSM5624634 Query DataSets for GSM5624634
Status Public on Aug 31, 2022
Title 3 day_rep2
Sample type SRA
 
Source name Intramuscular adipocytes
Organism Bos taurus
Characteristics tissue: Intramuscular fat
Treatment protocol When the adipocytes reached 90% confluence, the differentiation medium was used to induce differentiation, which was a complete medium containing 1 g/mL DEX(Sigma, USA) ,0.5 mM IBMX and 5 μg/ml insulin (Sigma, USA).After 48 hours, the medium was changed to a complete medium containing 5 μg/ml insulin. The differentiation medium was replaced every two days. 
Growth protocol Intramuscular adipocytes were grown in complete medium, which consisted of DMEM-F12 (Gibco, USA) with 15% FBS and 1% penicillin/streptomycin, at 37°C and 5% CO2, The growth medium was replaced every two days.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0. Approximately 10 ug of total RNA representing a specific adipose type was subjected to isolate Poly (A) mRNA with poly-T oligoattached magnetic beads (Invitrogen). Following purification, the poly(A)- or poly(A)+ RNA fractions is fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the protocol for the mRNA-Seq sample preparation kit (Illumina, San Diego, USA), the average insert size for the paired-end libraries was 300 bp (±50 bp). And then we performed the paired-end sequencing on an Illumina Novaseq™ 6000 at the (lc-bio, China) following the vendor's recommended protocol.
RNA libraries were prepared for sequencing using standard Illumina protocols
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Data processing Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm8 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
Reads Per Kilobase of exon per Megabase of library size (FPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Data filtering steps.Prior to assembly, the low quality reads(1,reads containing sequencing adaptors; 2,reads containing sequencing primer;3, nucleotide with q quality score lower than 20 )were removed .software CutAdapter,parameters -o 5 -p 100
Read alignment software,software HISAT ,version 2.0 ,parameters -l fr-firststrand -mi 20 -mx 500000 -p 2 -b dta -q phred33-quals -x 9
perform expression level for mRNAs by calculating FPKM.stimate the expression levels of all transcripts.software StringTie ,version 1.3.0,parameters -b dta -q phred33-quals.
Genome_build: Beta_vulgaris.GCF_000511025.2_RefBeet-1.2.2
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample
 
Submission date Oct 13, 2021
Last update date Aug 31, 2022
Contact name Xinhao Ma
E-mail(s) maxinhao@nwafu.edu.cn
Organization name Northwest A&F University
Street address No. 3, Taicheng Road
City Xianyang
State/province Shaanxi
ZIP/Postal code 712100
Country China
 
Platform ID GPL26012
Series (1)
GSE185850 Bovine intramuscular adipocytes RNA-seq
Relations
BioSample SAMN22247187
SRA SRX12593957

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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