title: 5'OH RNA seq of M. smegmatis: uninduced control 6h
design: 5 OH libraries were constructed as previously described (Schifano et al., 2014 Nat. Comm.). Briefly, three micrograms of the purified RNA from induced and uninduced cultures were digested with Terminator. After purification, the samples were phosphorylated using T4 PNK  and re-purified. 5 adapter was ligated using T4 RNA ligase 1. The adapter-ligated RNA samples were run in a 6% TBE-Urea PAGE gel, excised to remove unbound adapters and precipitated in isopropanol. The reverse transcription reaction was performed using Superscript IV and the degenerate primer (5-GCCTTGGCACCCGAGAATTCCANNNNNNNNN-3). The cDNA was run on a TBE-Urea gel and fragments between 80 and 500 nts were purified. The libraries were amplified in a PCR reaction with Phusion HF DNA Polymerase. The primers used were RP1 (5- AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA -3) and RPIX (5-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3). The amplified libraries between 150-450 bp were gel purified and sequenced.