characteristics.text: 2012-10-06T17:01:49Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T16:00:22Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA1532014
characteristics.characteristic: External Id
characteristics.text: Wellcome Trust Sanger Institute
characteristics.characteristic: INSDC center name
characteristics.text: 2012-10-06T17:01:49Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T16:00:22Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS179575
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-1309:polyA+_testis_7060321
characteristics.characteristic: Submitter Id
characteristics.text: frozen sample
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/OBI_0000922
characteristics.characteristic: bio source type
characteristics.text: Clontech
characteristics.characteristic: biosourceprovider
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: Human polyA+ RNAs were purchased from Clontech. The RNAs were isolated from tissue samples using a modified guanidium thiocynate extraction method followed by polyA+ RNA selection with two rounds of oligo(dT)-cellulose columns. We experimentally assessed three categories of gene models: (1) spliced models in which one primer could be placed within 75 nt of a junction and that will result in about half of the sequencing reads covering the junction ('Multi-span'); (2) spliced models in which this was unfeasible ('Multi'); and (3) monoexonic genes ('Mono'). In the case of 'Multi-span' primers, the 'junction primer' is positioned within an exon not more than 65 bp away from the targeted junction to ensure that sequencing reads will cross the junctions with a minimum of 10 nt, while the second primer maps within an adjacent exon. To increase the fraction of junctions we could possibly test, we then designed primer pairs further away from the junction ('Multi' primers). Primers were designed using Primer3. We used parameters minimizing the formation of primer dimers and maximizing primers 'stickiness'. Primer pairs were further filtered for mapping within repeat-regions and alternative priming within 30 kb in duplications and paralogous sequences (maximum of two tolerated mismatches). First-strand cDNA samples were prepared from human poly(A)+ RNAs (BD-Clontech) with the SuperScript III kit (Invitrogen). Amplifications were performed in a final volume of 12.5 uL with JumpStart REDTaq ReadyMix (Sigma-Aldrich) and a primer concentration of 0.4 uM in 384-well plates format on an automatized Evoware platform (TECAN) combined with a Tetrad2 thermocycler (Bio-Rad) that allows processing four plates in parallel. Because monoexonic amplification is sensitive to genomic DNA contaminations, monoexonic models were assessed by amplification of cDNA in which a dNTP analog was incorporated using the mRNA Selective PCR Kit (TAKARA).
characteristics.characteristic: description
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: testis
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000384
characteristics.characteristic: sex
characteristics.text: polyA+_testis_7060321
characteristics.characteristic: title