characteristics.text: LIBEST_023005
characteristics.characteristic: External Id
characteristics.text: SAMN00165554
characteristics.characteristic: External Id
characteristics.text: Shanghai Institute of Veterinary Research, Chinese Academy of Agricultural Sciences
characteristics.characteristic: INSDC center name
characteristics.text: 2008-04-17T00:00:00Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-10-11T17:46:02.170Z
characteristics.characteristic: INSDC last update
characteristics.text: live
characteristics.characteristic: INSDC status
characteristics.text: Generic
characteristics.characteristic: NCBI submission model
characteristics.text: Generic.1.0
characteristics.characteristic: NCBI submission package
characteristics.text: Total RNA was extracted from 7d schistosomula of Schistosoma japonicum and 42d adult schistosoma worms by Trizol reagent. The EST sequenced subtractive library of 7day hepatic schistosomulum. This was performed using the SuperSmart PCR cDNA Synthesis kit and PCR-selected cDNA Subtraction kit (Clontech) according to the manufacturer's protocols. Briefly, 1ug of total RNA from 7d schistosomula was used in reverse transcription with a specific oligo(dT) primer, and double-stranded cDNAs were synthesized by 18 cycles. SMART cDNA from adult mixed sex schistosome (Driver) and the schistosomula (Tester) was digested with Rsa I to generate short, blunt-ended double-strand cDNA. After phenol extraction and ethanol precipitation, Rsa I-digested Tester was ligated with Adaptor I and Adaptor2 at 5'end of each strand of the tester cDNA. The adaptor 1-ligated and the adaptor 2-ligated tester cDNAs were then separately hybridized with an excess of dr iver cDNA at 68oC for 8h after denaturation at 98oC for 90s. The two hybridization samples were mixed together without denaturation and hybridized at 68oC overnight with an excess of fresh denatured driver cDNA. The resulting mixture was diluted in 200 ul dilution buffer followed by two PCRs. In the primary PCR, primers against adaptor 1 and adaptor 2 were used to selectively amplify differentially expressed cDNA. And then the product of the first PCR was used as template in the second PCR with nested primers. The final PCR product was cloned into the pGEM-T vector (Promega) with blue and white selection in E.coli DH5a.
characteristics.characteristic: description
characteristics.text: 7d schistosomula
characteristics.characteristic: dev-stage
characteristics.text: Schistosoma japonicum
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_6182
characteristics.characteristic: organism
characteristics.text: Chinese mainland strain
characteristics.characteristic: strain
characteristics.text: Schistosoma japonicum 7d schistosomula subtractive expressed sequence tags library
characteristics.characteristic: title
characteristics.text: promega T easy vector
characteristics.characteristic: vector