title: Illumina HiSeq 2000 sequencing; GENCODE PCR-Seq Batch XVII
design: GENCODE PCR-Seq Batch XVII
library_construction_protocol: Human polyA+ RNAs were purchased from Clontech. According to the manufacturer, normal organ samples were pooled from a variable number of deceased individuals who had died of sudden death at a wide range of ages. RNA was isolated from these samples using a modified guanidium thiocynate extraction method, and enriched in polyA+ RNAs with two rounds of oligo(dT)-cellulose column purification. 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 Î¼L with JumpStart REDTaq ReadyMix (Sigma-Aldrich) and a primer concentration of 0.4 Î¼M 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).
attributes.tag: Experimental Factor: liver
attributes.value: organism part