Experiment SRX040787 Download

TitleTrabscriptome of Heliothis virescens
AliasH virescens Illumina GAII
Design descriptionmRNA was isolated from total RNA samples using oligo-d(T)25 magnetic beads (Dynabeads: Invitrogen, CA, USA). Once dynabead-mRNA binding is chemically reversed, the mRNA will be used as a template for 1st strand cDNA that will be converted to double stranded cDNA. The resulting double-stranded (ds) overhang fragments are end-repaired by incubation in the presence of T4 DNA polymerase and Klenow polymerase. The polished fragments are phosphorylated by T4 PNK, followed by the addition of a single ‘A’ base to the 3' end of the blunt-ended phosphorylated fragments. This ‘A’ base prepares the cDNA fragments for ligation to proprietary adapter oligonucleotides (Illumina single-read or paired-read sequences) which have a ‘T’ base at their 3' end. Ligation products are size-selected by gel electrophoresis and purification (2% low-range agarose with ethidium bromide). Following 1-2 hours at 80-110V at room temperature, the library range is visualized under brief UV and the 500bp band will be excised with a clean scalpel. Purified DNA libraries are subjected to a final PCR amplification step (15 cycles). All amplified libraries are quantitatively and qualitatively assessed by Nanodrop ND-1000 (Thermo Scientific, DE, USA) UV/Vis spectroscopy, DNA bioanalyzer 2100 (Agilent, CA, USA) microfluidics, and realtime quantitative PCR (Life Technology,CA, USA) to determine sequenceable molecules.
Referencesno content
OrganismsHeliothis virescens
SubmitterAgricultural Research Service, US Department of Ag
InstrumentIllumina Genome Analyzer II
Library nameHv_Illumina@NCGR
Library strategyEST
Library selectioncDNA
Library layoutSINGLE
Library construction protocolno content
Related objects
Linksno content