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Sample GSM2227366

Query DataSets for GSM2227366

Status

Public on Jul 10, 2016

Title

BZJ_5_T

Sample type

SRA

Source name

Longissimus thoracis

Organism

Capra hircus

Characteristics

sample source: ovariectomized goat
breed: Hybrid goats (Boer goat(male) and Guan Zhong dairy goat(female))
Sex: female
experimental treatment (ovariectomy) age: about 5 months old
slaughter age: after treatment 50th days

Treatment protocol

The goats in the treatment group were ovariectomized at the beginning of the experiment and the goats in control group were untreated. All the five goats were slaughtered at the 50th day.

Growth protocol

Five goats were feed on the same fodderand with similar body weight

Extracted molecule

polyA RNA

Extraction protocol

The longissimus thoracis were collected immediately after slaughtering, and were snap-frozen in liquid nitrogen, then stored at -80oC until RNA extraction.Total RNA was extracted from the sample using Trizol reagent (Takara, Dalian, China) according to the manufacturer’s instructions.
RNA samples with a RIN value greater than 8.0 (8.0 out of 10.0), OD260/ OD280 ratio greater than 1.9 and OD260/ OD230 ratio is no less than 1.7 were selected for deep sequencing.The RNA was treated with DNase I for 30 min at 37oC to remove residual DNA. Then enrich and purify mRNA using beads with oligo (dT).The purified mRNA was fragmented to approximately 350 nt fragments using the RNA fragmentation kit. The first strand cDNA was synthesized using purified RNA fragments as template and hexamer primers. After the first strand was synthesized, the buffer (Invitrogen, 20 μL), dNTPs (0.25 mM / μL), RNaseH (0.05 U / μL) and DNA polymerase I (0.5 U / μL) were added to synthesize the second strand cDNA (Zhang et al., 2015). The short cDNA fragments were further purified using the QIAQuick PCR extraction kit (LianChuan Sciences, Hangzhou, China). The samples were then subjected to 3’ end repaired, a single “A” base and an adaptor ligation. Agarose gel electrophoresis was used to filter the suitably sized fragments (350 ± 50 bp), which would be used as the templates for the PCR amplification and RNA-seq library generation. Then the sequencing of the libraries was performed using Illumina HiSeq 2000 (LianChuan Sciences, Hangzhou, China).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer

Description

treatment group

Data processing

Illumina CASAVA version1.8+ soft were used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence. The transcript and unigene assembly was carried out using the short assembly program Trinity methodthen (MG Grabherr, 2011).
The gene expression level was computed by bowtie 0.12.8 soft using single-end mapping method, and further measured by the Reads Per Kilobase of exon model per Million mapped reads value (RPKM), which can be calculated as follow: RPKM=Total exon reads/[mapped read (millions) * exon length (KB)] (Mortazavi et al., 2008).
All assembled unigenes were compared with public database, NCBI non-redundant protein sequences (NR), SWISS-PROTProt, Kyoto Encyclopedia of Genes and Genomes (KEGG), euKaryotic Ortholog Groups (KOG) and Pfam, to make functional annotation by similarity analysis. Sequences alignment using BLAST software.
Genome_build: De novo assembly
Supplementary_files_format_and_content: tab-delimited text files

Submission date

Jul 06, 2016

Last update date

May 15, 2019

Contact name

Sihuan Zhang

E-mail(s)

sihuanzhang1990@163.com

Phone

18700808486

Organization name

Northwest A&F University