characteristics.text: LIBEST_020836
characteristics.characteristic: External Id
characteristics.text: SAMN00152928
characteristics.characteristic: External Id
characteristics.text: Universita Cattolica S. Cuore
characteristics.characteristic: INSDC center name
characteristics.text: 2007-02-06T00:00:00Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-10-11T17:45:01.150Z
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: Biondo cadenera,Tarocco nucellare 57-1E-I,Moro nucellare 58-8D-I
characteristics.characteristic: cultivar
characteristics.text: Fruits were harvested at three developmental stages. Poly-A mRNA was isolated from total RNA extracted from flesh according to the Trizol LS and Dynabeads M-280 Streptavidin protocols. A cDNA-AFLP technique of RNA fingerprinting was optimized allowing the detection of DNA fragments derived from RNA using cDNA synthesis and subsequent polymerase chain reaction (PCR) amplification. This is based on the selective amplification of cDNA fragments obtained by digestion with one frequent and one rare cutter enzymes, followed by ligation of two double-stranded adapters of known sequences. AFLP-TP (Transcriptional Profiling) is an improved version of the cDNA-AFLP protocol, that allows the isolation one unique restriction fragment for each cDNA. The 'esacutter' enzyme is BstYI (recognition site Pu/GATCPy) and the 'fourcutter' is MseI (recognition site T/TAA). The difference in AFLP-TP lies in an additional step between the two digestions: as the cDNA synthesis is obtained with a biotinylated oligo-dT, this allows to collect the 3' end of the transcript after the first digestion, taking advantage of the high affinity of the biotin for the streptavidin anchored to magnetic beads (Dynal). After the electrophoretic run, a vertical scanner collected the fluorescence emissions derived from the excitation of the fluorescein molecules attached to the BstYI primers with a 465nm monochromatic beam. Single fragments were isolated either through DGGE (Denaturing Gradient Gel Electrophoresis) analysis or cloning into pGEM-T Easy vector (Promega). Samples that showed only one neat band were sequenced.
characteristics.characteristic: description
characteristics.text: three developmental stages during ripening period
characteristics.characteristic: dev-stage
characteristics.text: Citrus sinensis
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_2711
characteristics.characteristic: organism
characteristics.text: BstYI
characteristics.characteristic: re_1
characteristics.text: MseI
characteristics.characteristic: re_2
characteristics.text: flesh
characteristics.characteristic: tissue-type
characteristics.text: Citrus sinensis flesh cDNA-AFLP Library
characteristics.characteristic: title
characteristics.text: Plasmid
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/SO_0000155
characteristics.characteristic: v_type
characteristics.text: PGEM Teasy vector
characteristics.characteristic: vector