RIP-seq of HA-tagged Srb in M. smegmatis (log)
Total RNA was extracted using bead beating in lysis buffer [20 mM Tris-HCl pH 7.5, 150 mM KCl, 1 mM MgCl2, 1 mM DTT, 1 mM PMSF, 0.2% Triton X100, 20 U/ml DNase I (Thermo Scientific), 200 U/ml SUPERase-IN (Life Technologies). Co-immunoprecipitation was performed using anti-HA-Tag (6E2) mouse monoclonal antibody (NEB) and Protein G Dynabeads (Thermo Scientific). The sample was dephosporylated and the cDNA library was constructed using the NEBNext small RNA library prep set for Illumina (NEB). The 150-400 bp region was selected according to a 6% polyacrylamide gel and sent for sequencing on the 1x100 mode.