Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells
Total RNA was isolated from cell pellets treated with RNAlater using the RNA mini kit (Qiagen, Valencia, CA) and treated with DNaseI (Qiagen, Valencia, CA) for 30 min at room temperature. Following ethanol precipitation, biotinylated LNA oligonucleotide rRNA probes complementary to the 5S, 5.8S, 12S, 18S and 28S ribosomal RNAs were used to deplete the rRNA from 5 µg of total RNA by RiboMinus (Life Technologies, Carlsbad, CA) as per manufacturer's instructions. Purified RNA (50 ng) was fragmented by metal hydrolysis in 1X fragmentation buffer (Life Technologies, Carlsbad, CA) for 15 min at 70 °C, stopping the reaction by addition of 2 µl fragmentation stop solution (Life Technologies, Carlsbad, CA). Fragmented RNA was used to generate strand-specific RNA-seq libraries as per the Directional mRNA-seq Library Preparation Protocol (Illumina, San Diego, CA).