Trypanosoma vivax RNAseq (BF)_ Illumina
Library construction and sequencing.  Double-stranded cDNA was generated from 25 µg of total RNA using a SuperScript II Double-Stranded cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s instructions, except for oligonucleotide used for first strand synthesis and 5-methyl-dCTP (Jena Biosciences) instead of dCTP. The primer used was 5’ CTGGAG(T)16VN 3’, the 5’ end of the primer contain the restriction site for the enzyme GsuI. After the synthesis of the second strand, the cDNA was precipitated with 1/10 volumes of Sodium Acetate (3M, pH =5.2), 2 µL of glycogen (15 µg/mL) and 3 volumes of absolute ethanol and resuspended in 70 µL of RnaseFree water. 65 µL of cDNA was digested with GsuI (Fermentas) for 4 hours at 30°C to cleave the poliA tails. The digested cDNA was used to prepare the 454 and Illumina libraries.  454 library preparation and sequencing.  We prepared the library with the GS Titanium DNA library preparation kit (Roche) according the manufacturer’s protocol starting with 2.5 µg of cDNA. The emPCR was done with GS Titanium SV empPCR kit (Roche) according with manufacturer protocol. We use GS Titanium Sequencing Kit XLR70 (Roche) to sequenced 1/2  GS Titanium PicoTiterPlate kit 70x75 in 454 Genome Sequencer FLX System (Roche).  Illumina sequencing was carried out in a GAIIx on the same cDNA library that was re-fragmented and universal Illumina adaptors were added.