title: Transcription of Calmodulin locus in Trypanosoma cruzi
design: We used a unidirectional amplicon sequencing approach to sequence the 5'UTR of calmodulin gene from different stages of T. cruzi strain Y and CL-Brener. To do so we engined an RT-PCR using sense primers with a specific bar-coding for each stage of strain Y and the epimastigote stage of CL-Brener, a sequence of the primer A of the library L (Lib-L) and a sequence specific for mini-exon gene of T. cruzi as well as an antisense primer with a sequence specific for a very conservate region of the calmodulin 3'UTR and a specific sequence of primer B of Lib-L. The amplicon library was prepared following the workflow of the Amplicon Library Preparation Method Manual for the 454GS Junior Titanium. The PCR was carried out using 1 unit of taq polimerase platinum (Life technologies, Grand Island, USA), 2 mM of Magnesium Chloride,  200µM of dNTPs,  5µl of 10X buffer, 100 nM of primer sense and anti-sense and water to complete 50 µl. The PCR was performed at 94°C for 2 minutes follow by 35 cycles of 97°C for 15 seconds, 60°C for 20 seconds, 72°C for 30 seconds and a final extension of 72°C for 7 minutes. All products were visualized in agarose gels stained with GelRedTM Nucleic Acid gel stain (Biotium Inc, Hayward, USA). The amplicons were purified using the commercial kit Agencourt AMPure XP - PCR Purification (Beckman Coulter, USA) following manufacturer instructions and the quantification was performed using the fluorimetric assay Quant-iT PicoGreen dsDNA Assay Kit (Life technologies, Grand Island, USA).  The emulsion -PCR and pyrosequencing were carried out at the sequencing facility of the program of technological development and health supplies (PDTIS) of the Instituto Oswaldo Cruz following instruction of the emPCR Amplification Method Manual- Lib-L and Sequencing Method Manual for 454 GS Junior, respectively.