title: Pseudouridine RNA modification on small non-coding RNAs in Trypanosoma brucei : identification of novel sites  and  functional implications
design: RNA (~800 ng) was fragmented and dephosphorylated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific), cleaned by Agencourt RNA clean XP beads (Beckman Coulter) and ligated to a 3 linker using high concentration T4 RNA Ligase 1 (NEB) in a buffer containing DMSO, ATP, PEG 8000 and RNase inhibitor (NEB). The ligated RNA was cleaned from excess linker using Dynabeads MyOne SILANE beads (Thermo Scientific), and first strand cDNA was prepared using the TGIRTTM-III Enzyme (InGex). The RNA was subsequently degraded using 2 ml of 1 M NaOH and the cDNA was cleaned using Dynabeads MyOne SILANE beads (Thermo Scientific). The cDNA was further ligated to a 3 adapter using high concentration T4 RNA Ligase 1 (NEB) 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) and sequenced in a Nextseq system (Illumina) in paired end mode (40 million reads for each sample).