RNA-seq of Trypanosoma brucei WT sample rep 1
For poly (A) RNA preparation total RNA was incubated with Dynabeads mRNA DIRECT (Invitrogen) as per manufacturers instruction. Poly (A) 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 AffinityScript Reverse Transcriptase enzyme (Agilent) or TGIRTTM-III Enzyme (InGex). The RNA was subsequently degraded using 2 l 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 enriched by PCR using NEBNext High-Fidelity (NEB) polymerase (9 PCR cycles) and sequenced in