Rneasy mini cleanup

MOE^LOW (passages 8, 9, and 10) and MOE^HIGH (passages 90, 103, and 113) cells were plated and at 75% confluency, they were washed with PBS, lysed, and total RNA extracted in Trizol reagent (Life Technologies), according to manufacturer’s protocol. To ensure that there was no residual genomic DNA contamination, samples were treated with RNase-free DNase I and RNA was further purified using RNeasy Mini cleanup (Qiagen Inc., Valencia, CA, USA). The concentration of the total RNA was determined using the NanoVue Plus spectrophotometer provided by the UIC Resource Research Center Core (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) and RNA purity as determined by the RNA integrity number (RIN) was verified using TapeStation 2200 (Agilent Technologies, Palo Alto, CA, USA).
Each total RNA sample had ribosomal RNA removed using TruSeq Stranded Total RNA with Ribo-Zero (Illumina, San Diego, CA, USA). Strand-specific libraries were constructed and quantitated using Qubit, and cDNAs verified by qPCR. The resulting six libraries were pooled and sequenced using TruSeq SBS sequencing kit 3 for 101 cycles on a HiSeq2000 (Illumina) and processed with Casava (version 1.8.2.), according to the manufacturer’s protocol.