Global Transcript Profiling of Sulphur Metabolism in Cotyledon Stage Seeds of Vigna Mungo and P.vulgaris via 454 cDNASequencing
High quantity and quality of total RNA was isolated from V.mungo seeds containing phenolic compounds and BAT93 using the basic RNA extraction buffer with the addition of  BSA, heparin, Proteinase K and PVPP (Wang et al. 1994).480µg of DNaseI treated-total RNA after  acidic Phenol: chloroform purification to remove DNase I was proceeded for enrichment of messenger RNA and depletion of ribosomal RNA  following the protocols using Poly(A)PuristTM  MAG Magnetic mRNA Purification Kit(Ambion AM1922) and then RiboMinus™ Plant Kit (Invitrogen). 400ng of enriched mRNA was used for Roche 454 rapid cDNA library construction following 2009 October version of cDNA Rapid Library Preparation Method Manunal(Rev. Jan 2010). Following cDNA rapid library preparation, 3 copies per bead (cpb) were used for emPCR and then 2x106  beads were loaded per half plate for 454 pyrosequencing  on  Genome Sequencer (GS) FLX Titanium platform using 2009 October version  sequencing manual(Rev Nov 2010) of 454 Titanium chemistry(Roche Applied Sciences)