Total RNA was isolated from cell pellets treated with RNAlater using the RNA mini kit (Qiagen) and treated with DNaseI (Qiagen) for 30 min at room temperature (22 °C). After ethanol precipitation, biotinylated LNA oligonucleotide ribosomal RNA (rRNA) probes complementary to the 5S, 5.8S, 12S, 18S and 28S rRNAs were used to deplete the rRNA from 5 μg of total RNA by RiboMinus (Life Technologies) as per the manufacturer's instructions. Purified RNA (50 ng) was fragmented by metal hydrolysis in 1× fragmentation buffer (Life Technologies) for 15 min at 70 °C, stopping the reaction by addition of 2 ml fragmentation stop solution (Life Technologies). Fragmented RNA was used to generate strand-specific RNA-Seq libraries as per the Directional mRNA-Seq Library Preparation Protocol (Illumina).
