characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2014-02-11T17:00:59Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T16:58:44Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2266663
characteristics.characteristic: External Id
characteristics.text: Centre For Research in Agricultural Genomics (CRAG)
characteristics.characteristic: INSDC center name
characteristics.text: 2014-02-11T17:00:59Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T16:58:44Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS374066
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-1868:43TG
characteristics.characteristic: Submitter Id
characteristics.text: 4 week
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UO_0000034
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: pig
characteristics.characteristic: common name
characteristics.text: Protocols: Cells were grown at 37C and 5% CO2 and maintained in Dulbeccos Modified Eagle Medium (DMEM) supplemented with 5% FCS, 100 ug/ml streptomycin and 100 IU/ml penicillin. A viral stock with a titre of 107,57 TCID50/mL was prepared in PK-15 cells for NIA-3 virulent strain and 108,49 TCID50/mL for Begonia attenuated strain. In the in vivo experimental infection, 9 animals were intranasally inoculated with 103 TCID50 NIA-3 virulent strain, 6 with the same dose of Begonia attenuated strain and 5 control animals with PBS. Total RNA was isolated using TRIzol reagent. Small RNA fraction from each sample was excised and isolated from denaturing 12.5% polyacrilamide gel electrophoresis (PAGE) using miSpikeTM (IDT, Coralville, USA) as internal size marker. 50 ug of total RNA for each sample were loaded on separate gels to avoid cross-contamination. Gels were stained with GelStar Acid Nucleic Gel Stain (Lonza, Basel, Switzerland) for UV visualization. Excised small RNA fraction were purified using Performa DTR gel filtration cartridges (EdgeBio, Gaithersburg, USA). Briefly, 3' and 5' linkers from miRCatTM kit (IDT, Coralville, USA) were ligated at both ends of the small RNAs in two separated reactions using a T4 RNA ligase without ATP (Fermentas, Germany) and T4 RNA ligase with ATP (Ambion, Austin, USA), respectively. Between 3' and 5' primer ligations, the 60 nt RNAs were purified by PAGE to eliminate unligated products. Then, linked products were used to perform a reverse transcription reaction using the SuperScriptTM III Reverse Transcriptase kit (InvitrogenTM, Carlsbad, USA) and the cDNA obtained was amplified with the Expand High Fidelity System (Roche, Germany). PCRs were done to amplify the cDNA with primers complementary to 3' and 5' linkers and, in addition, they included multiplex identifiers at the 5' end (a five nucleotide sequence tag) to allow differentiation between libraries. The number of PCR cycles was optimized for each sample, in order to minimize/avoid saturation, ranging from 21 to 30. Purification was carried out by using QIAquick PCR Purification Kit (Qiagen, Germany). Libraries were quantified with QubitTM fluorometer, Quant-ITTM (InvitrogenTM, Carlsbad, USA), prepared to a 1011 DNA molecules/uL and equimolecular pooled according to their indexes.
characteristics.characteristic: description
characteristics.text: Begonia infected
characteristics.characteristic: disease
characteristics.text: 43TG
characteristics.characteristic: individual
characteristics.text: Sus scrofa
characteristics.characteristic: organism
characteristics.text: Sus scrofa
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9823
characteristics.characteristic: organism
characteristics.text: Sus scrofa
characteristics.characteristic: scientific_name
characteristics.text: frozen specimen
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/OBI_0000922
characteristics.characteristic: specimen with known storage state
characteristics.text: 43TG
characteristics.characteristic: title