For total RNA isolation, a frozen pellet of cells was resuspended in 1 ml TRIzol reagent (Life Technologies) to homogenization and incubated at room temperature for 5 min.  To the resuspension, 0.2 ml chloroform was added and mixed by inverting the tube for 15 seconds. The mixture was incubated at  room temperature for 10 min and then centrifuged at 20,000 g for 10 min at 4°C.  After centrifugation, ~0.6 ml of the upper phase was transferred to a new centrifuge tube containing 0.5 ml isopropanol.  The mixture was incubated at room temperature for 10 min and then centrifuged at 20,000 g for 15 min at 4°C.  After centrifugation, the supernatant was discarded and the pellet was washed twice with 1 ml 75% ethanol, made with Diethyl pyrocarbonate (DEPC) treated water, by centrifugation at 12,000 rpm, for 5 min at 4°C. After the second wash, the tube was left open for 10-15 min at room temperature to dry the pellet. To the pellet, 50 µl DEPC treated water was added and the tube was left at 37°C for 10-15 min and then the pellet was fully resuspended using a pipette.  DNA was removed using TURBO DNA-free™ Kit (Invitrogen). Quality of total RNA was determined by electrophoresis on the TapeStation system (Agilent). Paired-end sequencing libraries were prepared with 2.5 µg of total RNA using Illumina® TruSeq® Stranded Total RNA library prep workflow with Ribo-Zero according to the manufacturer's instructions.
GEO Accession
GSM3963055