|
| Status |
Public on Jun 18, 2012 |
| Title |
longissimus muscle_pooled 3 Lantang postnatal 180 day pigs |
| Sample type |
SRA |
| |
|
| Source name |
pooled longissimus muscle of three pigs (postnatal day 180)
|
| Organism |
Sus scrofa |
| Characteristics |
breed: Lantang
tissue: longissimus muscle
developmental stage: postnatal day 180
|
| Treatment protocol |
Purebred 15 Lantang (LT) and 15 Landrace (Lde) sows which have the same genetic background were artificially inseminated with semen from same purebred boars respectively.
|
| Growth protocol |
All animal procedures were performed according to guidelines developed by the China Council on Animal Care and protocol approved by Animal Care and Use Committee of Guangdong Province, P.R. China.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The RNA library was constructed and deep sequencing preformed by the Beijing Genomics Institute (BGI, commercial service). Total RNA was extracted from frozen muscles using standard protocols (Trizol) and then treated with DNase to remove potential genomic DNA contamination according to the manufactures's protocols. RNA integrity and concentration were evaluated by Agolent 2100 Bioanalyzer (Agilent Technologies). For RNA library construction and deep sequencing, equal quantities of RNA samples from three individual pigs were pooled at every time points. Approximately 6 ug of RNA representing each group were submitted to Solexa (now Illumina Inc.) for sequencing. Sequence tag preparation was done with Illumina's Digital Gene Expression Tag Profiling Kit according to the manufacturer's protocol. In brief, mRNA was isolated from 6ug total RNA by binding the mRNA to a magnetic oligo bead. First-and second-strand cDNA were synthesized while the mRNA was attached to the beads. The double 14 stranded cDNA were digested with NlaIII to wash away all fragmens other than the 3? CATG fragment attached to the oligo bead. Then GEX NlaIII Adapter 1 was ligated at the site of NlaIII cleavage. In addition, GEX NlaIII Adapter 1 contains the sequence for the restriction enzyme MmeI, subsequently, we applied the restriction enzyme MmeI to create the 17 bp tag. The GEX Adapter 2 was ligated at the site of MmeI cleavage. A PCR with 12 cycles was performed with two primers that anneal to the ends of the adapters to enrich the adapter-ligated cDNA construct. The resulting 85 bp fragments were purified from 6% Novex TBE PAGE gel. Subsequently, the purified cDNA tags were sequenced on the Illumina Cluster Station and Genome Analyzer. Image recognition and base calling were performed using the Illumina Pipeline.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Data processing |
Data processed by the Beijing Genomics Institute (BGI, commercial service). Clean tags were obtained by filtering raw data to remove adaptor tags, low quality tags and tags of copy number = 1. The clean tags were classified according their copy number in the library and the proportion of each category in relation to total clean tags was determined. Similar analyses were carried out for clean distinct tags.
All possible CATG+17-nt tag sequences were created from the sus scrofa RefSeq and UniGene (NCBI36.1, 20090827) databases and used as reference sequences to align and identify the sequencing tags. All clean tags were aligned to the reference database, and then unambiguous tags were annotated. One mismatch in each alignment was allowed to tolerate polymorphisms across samples. Mismatch could be caused by a sequencing error, but the frequency is very low (1 or 2 per million).
Software/programs used in our analysis:
1. BLAST v2.2.21. (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.2.21/)
2. BLAT v.34, (http://genome.ucsc.edu/FAQ/FAQblat)
3. SOAP v1.11. . http://soap.genomics.org.cn/#down2.
4. Bowtie v0.12.3. http://bowtie-bio.sourceforge.net/index.shtml
5. Cluster v1.47. http://bonsai.ims.u-tokyo.ac.jp/~mdehoon/software/cluster/
6. Java TreeView v1.1.4r3. http://sourceforge.net/projects/jtreeview/
7. Medusa v1.5.1. http://coot.embl.de/medusa/ Applet. http://coot.embl.de/medusa/Stringlet2Applet.shtml
8. Blast2GO Blast2GO. v2.3.5. http://www.blast2go.org/
|
| |
|
| Submission date |
Nov 16, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Jason Karpac |
| E-mail(s) |
karpac@tamu.edu
|
| Organization name |
Texas A&M University Health Sciences Center
|
| Department |
Molecular and Cellular Medicine
|
| Street address |
Reynolds Medical Building
|
| City |
College Station |
| State/province |
Texas |
| ZIP/Postal code |
77843 |
| Country |
USA |
| |
|
| Platform ID |
GPL9126 |
| Series (1) |
| GSE25406 |
Comparative analyses of transcriptome during skeletal muscle development between pig breeds differing in muscle growth rate by deep-sequencing |
|
| Relations |
| SRA |
SRX054608 |
| BioSample |
SAMN00254192 |
