GSM5664501: PRS4; Trypanosoma brucei brucei TREU927; OTHER
Whole cell extracts were prepared from 5x10^9 T. brucei cells in a buffer containing 24 mM KCl, 20 mM Tris-pH7.8 and 10 mM MgCl2. RNPs were extracted with 300 mM KCl, and the ribosomes were removed by centrifugation for 3 hr at 35,000 rpm in a Beckman 70.1Ti rotor (150000 x g). The supernatant containing the post-ribosomal supernatant (PRS) was deproteinized by digestion with proteinase K (Roche) (100 ug/ml in 1% SDS for 30 min at 37oC), and RNA was prepared using TRIzol (Sigma) reagent. Libraries were created following the updated RibOxi-seq protocol (https://link.springer.com/protocol/10.1007%2F978-1-0716-1851-6_22). Fragmentation step was modified to use 250ng small RNA for benzonase digestion with fragmentation of 30 minutes instead of the much longer reaction time the original protocol calls for.