Journal: BMC Genomics
Article Title: A platform independent RNA-Seq protocol for the detection of transcriptome complexity
Figure Lengend Snippet: Schematic representation of the method. A . Library preparation, starting from deprived ribosomal fraction of total RNA. The two strand-specific tags, rv5-3 and complementary fw5-3 tag (respectively in grey and black) are represented. a) Synthesis of the 1 st cDNA strand, using the Tag-random-octamer primer rv5-3tag. b) 1 st strand cDNAs ligation. c) Synthesis of the 2 nd cDNA strand with random primers and Phi29 DNA polymerase amplification. This step produced the fw5-3 tag. d) Mapping of the reads using strand-specific tags to correctly assign reads onto the genome. If the gene x is on the positive strand (+) of the genome, whereas the gene y on the negative strand (-), reads tagged with fw5-3 tag map gene x on strand + and gene y on strand -. Conversely rv5-3 tagged reads map an opposite orientation in the same locus. B . Removal of ribosomal RNAs from total RNAs. Efficiency of rRNA depletion was evaluated by means of an Agilent 2100 Bioanalyzer using the Agilent RNA 6000 Nano Kit. Both in the OST-78 and in the OST-83 samples, a dramatic reduction in 18S and 28S rRNA bands compared to the pre-Ribominus samples was confirmed. C . OST-78 and OST-83 RNA samples were retro-transcribed, ligated and amplified with the Phi29 DNA polymerase. After 4 h amplification, we obtained DNA fragments of high molecular weight (about 23 kb) visualized on 1% agarose gel.
Article Snippet: The removal of the ribosomal component from the total RNA was performed using the RiboMinus Eukaryote Kit for RNA Seq (Invitrogen), according to the manufacturer’s instructions.
Techniques: Ligation, Amplification, Produced, Molecular Weight, Agarose Gel Electrophoresis