After treatment, cells were washed with PBS and lysed with TRIzol reagent (Invitrogen) The first step is selectively purifying the poly-A containing mRNA molecules using oligo-dT attached magnetic beads. Following purification, the mRNA is fragmented using divalent cations under elevated temperature. Next, the cDNA is synthesized using Reverse transcriptase and random hexamers in a first strand synthesis reaction. Subsequently, the cDNA is converted to double stranded cDNA where Uracil is added instead of Thymine. The strand specificity is preserved by a USER enzyme based digestion of the second strand thereby leaving one functional strand which maps to the DNA strand which it was transcribed from. The USER digested single strand molecules are enriched and indexed in a limited cycle PCR followed by AMPure bead purification to create final cDNA library for sequencing.