|
|
GEO help: Mouse over screen elements for information. |
|
Status |
Public on Aug 31, 2022 |
Title |
0 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 |
SAMN22247183 |
SRA |
SRX12593954 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
|
|
|
|
|