title: RiboMethSeq of T. brucei: Grumpy OE RNA
design: Trypanosoma brucei parasites were grown in SDM-79 medium supplemented with 10% fetal calf serum at 27oC. Total RNA from T. brucei PCF and BSF was denatured at 90oC for 2 min in a thermocycler.  Then, an equal volume of buffer (NaHCO3/Na2CO3, pH 9.9) was added, and RNA samples were incubated at 90oC for 20 minutes. The hydrolyzed RNA was then used for library preparation. Briefly, 800ng of the RNA was dephosphorylated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific) and cleaned by Agencourt RNA clean XP beads (Beckman Coulter). The RNA was then ligated to 3 linker using high concentration T4 RNA Ligase 1 (NEB) in a buffer containing DMSO, ATP, PEG 8000 and RNase inhibitor (NEB) for 1.5hr at 22oC.  The ligated RNA was purified from excess linker using Dynabeads MyOne SILANE beads (Thermo Scientific), and first strand cDNA was prepared using the AffinityScript Reverse Transcriptase enzyme (Agilent) at 55oC for 45 min. Next, the RNA was degraded using 2ul of 1M NaOH, and the cDNA was cleaned using Dynabeads MyOne SILANE beads (Thermo Scientific). The cDNA was further ligated to 3 adapter using a high concentration T4 RNA Ligase 1 (NEB) overnight at 22oC and cleaned of excess adapter by using Dynabeads MyOne SILANE beads (Thermo Scientific). The adapter ligated cDNA was PCR enriched using NEBNext High-Fidelity (NEB) polymerase (9 PCR cycles), separated on an E-Gel EX agarose gel (Invitrogen) and size selected for the range of 150-300 bp (containing ~30-180nt corresponding to RNA). The amplicons were gel purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel), and sequenced in a Nextseq system (Illumina) in paired end mode (20-40 million reads for each sample).